Human Molecular Genetics, 2002, Vol. 11, No. 24 3107-3114
© 2002 Oxford University Press
Rescue of neurodegeneration in Niemann–Pick C mice by a prion-promoter-driven Npc1 cDNA transgene
1National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA, 2Angel Charity for Children—Wings for Genetic Research, Steele Memorial Children's Research Center, Department of Pediatrics, University of Arizona, Tucson, AZ, USA, 3Department of Molecular and Cellular Biology and Genetics Committee, University of Arizona, Tucson, AZ, USA, 4Sidney Weisner Laboratory of Genetic Neurological Disease, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA and 5Diagnostic and Research Services Branch, Veterinary Resources Program, Office of Research Services, National Institutes of Health, Bethesda, MD, USA
Received August 19, 2002; Accepted September 23, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Niemann–Pick disease type C (NPC) is a neurodegenerative disorder with major visceral complications, including liver disease that can be fatal before onset of neurodegeneration. We have sought to determine the extent to which visceral disease contributes to neurodegeneration by making transgenic mice in which the wild-type NPC1 protein is expressed primarily in the CNS using the prion promoter. When the transgene was introduced into the npc1-/- animals neurodegeneration was prevented, a ‘normal’ lifespan occurred and the sterility of npc1-/- mice was corrected. The rescue did not provide complete neurological correction in the CNS as GM2 and GM3 gangliosides were observed to accumulate in some neurons and glia of transgenic animals. Two of three transgenic lines demonstrated some low-level ectopic expression resulting in correction of visceral phenotypes in liver and spleen. Interestingly, the third transgenic line continued to have moderate histocytosis in liver and spleen, yet had no detectable neurodegeneration. Thus, it is primarily the lack of NPC1 in the CNS and not the secondary effects of the visceral involvement that causes the neurological decline in NPC disease. In addition, the expression levels of NPC1 found in the CNS of transgenic animals were much greater than in normal littermates, demonstrating that overexpression of NPC1 is not harmful and allowing possibilities for genetic therapy interventions that utilize overexpression.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Niemann–Pick disease type C (NPC) is an autosomal recessive neurodegenerative disorder characterized by intracellular accumulation of unesterified cholesterol and gangliosides within the endosomal–lysosomal system (1). Patients present with both visceral defects of hepatosplenomegaly and neurological degeneration involving ataxia, dystonia, seizures and vertical supranuclear gaze palsy (2). This disorder is genetically heterogeneous with two complementation groups (3,4). The first NPC1 locus accounts for 95% of the individuals and has been identified by mutations in the Npc1 gene (5,6). The second locus, NPC2, has only recently been identified as the lysosomal protein HE1 (7).
Identification of the major gene responsible for the disorder, Npc1, revealed a multi-pass transmembrane protein containing a sterol-sensing domain that shows homology to the genes Patched, HMG CoA Reductase and SREBP Cleavage Activating Protein (5,6). Analysis of NPC1 protein function suggests that it is involved in late endosomal lipid sorting/vesicular trafficking (8–10), or may function as a transmembrane efflux pump (11). However, it remains to be elucidated what the precise function of the NPC1 protein is within the cell and why alteration of NPC1 results in the pathological changes observed in individuals with NPC.
A murine Npc1 animal model, Npc1N (referred to as npc1-/-), arising from a retroviral insertion in the Npc1 gene (6) has been a useful tool in the identification of the disease gene and in characterization of the cellular defects resulting from the disorder (12). The homozygous npc1-/- mice also present with progressive wasting, ataxia, hepatosplenomegaly and neurodegeneration, culminating in death of the mice between 8 and 11 weeks of age (13). Studies in the mouse model of NPC demonstrated that the accumulated cholesterol in liver was derived from low-density lipoprotein (LDL) (12). NPC mice exhibit massive accumulation of unesterified cholesterol in visceral tissue but for some time it was thought that the brain was free of such cholesterol storage (1,14). Recent studies, however, show that the apparent lack of accumulation of cholesterol in the brain is due to the loss of myelin which is rich in cholesterol (15) and that cholesterol levels are elevated in neurons (16). However, the relative importance of cholesterol storage in the soma versus the CNS is not clear.
In addition to cholesterol accumulation in visceral tissues, individuals with NPC also demonstrate CNS accumulations of other lipid molecules including glucosylceramide, lactosylceramide and complex gangliosides GM2 and GM3 (1,16). Recent studies suggest that cerebellar degeneration, visceral organ defects and viability are not rescued in npc1-/- mice that cannot synthesize GM2 and other complex gangliosides (17). In this paper, mice doubly null for Npc1 and ß1-4 GalNAcT, a transferase needed for the generation of GM2 and higher-order gangliosides, were examined for potential correction of NPC traits. Double mutant mice exhibited reduced vacuolization in the cerebral cortex consistent with reduced GM2 accumulation, yet a small number of foamy macrophage/microglial cells remained, suggesting that other molecules continued to accumulate. Purkinje cells, in contrast to most cortical neurons, underwent degeneration similar to npc1-/- mice. The lack of ß1-4 GalNAcT was found to have no effect on the visceral organ pathology, leading to the suggestion that CNS dysfunction in NPC might be secondary to effects of the visceral organ storage abnormalities.
In order to separate the CNS and visceral effects on ataxia and viability we have introduced into npc1-/- mice the expression of a wild-type murine Npc1 gene under the control of the mouse prion promoter. The prion promoter was selected based upon its ability to direct expression of a transgene primarily to CNS-derived tissues (18) with high levels in brain, intermittent levels in lung and low levels in spleen (19,20). We find that the characteristic phenotypes of wasting, ataxia, neurodegeneration, sterility and early fatality present in npc1-/- mice were rescued by the introduction of an Npc1 transgene.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A transgenic expression cassette was generated that expressed the murine Npc1 gene under control of the prion promoter (Fig. 1A). This transgenic construct Tg(Npc1) was used to generate three lines of transgenic mice Tg(Npc1)B2, Tg(Npc1)C2 and Tg(Npc1)C3. Each line was mated to heterozygous npc1+/- mice. The resulting transgenic npc1+/- F1 progeny were backcrossed to npc1+/- animals to generate npc1-/-, Tg(Npc1) mice. These transgene-positive, homozygous mutant mice were then compared with the original npc1-/- mice for analyses. Each of the independent lines was evaluated to determine if the transgene would be able to rescue the phenotypes of wasting, ataxia, neurodegeneration, hepatosplenomegaly, sterility and lethality that are characteristic of the npc1-/- mice model.
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Weight loss in npc1-/- mice occurs rapidly from 6 weeks until death at 8–11 weeks. Introduction of the transgene into the npc1-/- mice resulted in complete correction of the wasting phenotype observed in npc1-/- mice (Fig. 1B) and all animals remained viable for the 18 months during which the animals were observed. For npc1-/- mice, the rapid weight loss at week 6 is concurrent with the visible onset of the ataxic movements. The resulting ataxic tremors result in a shortened and staggering gait in npc1-/- mice (Fig. 1C). Tremors become progressively worse in these animals until death. No tremors were observed in mice from all three transgenic lines (Fig. 1C) for the 18 months of observation. Abnormal gait also was rescued in all three lines of transgenic mice. In comparison to wild-type mice, homozygous npc1-/- animals had a stride length 39% of wild-type stride length. Transgenic stride lengths were 94, 84 and 82% for npc1-/-, Tg(Npc1)C3; npc1-/-, Tg(Npc1)C2; and npc1-/-, Tg(Npc1)B2, respectively, in comparison to wild-type mice.
Male and female sterility was rescued in these mice for the two lines tested. For the C2 line, a pair of npc1-/-, Tg(Npc1)C2 mice were mated and 19 pups were delivered in the first 62 days. For the C3 line, a npc1-/-, Tg(Npc1)C3 female was mated to a npc1+/-, Tg(Npc1)C3 male and 14 pups were delivered in 65 days; and the same cross with sexes reversed yielded 14 pups in 78 days. While these numbers of pups are slightly reduced compared with those for young npc1+/- pairs (about 16 pups/60 days), they clearly demonstrate the fertility of two of the transgenic lines. The Tg(Npc1)B2 line was not tested.
The prion promoter directs expression of a transgene primarily to CNS-derived tissues (18). In order to evaluate the level of transgene expression in the CNS, northern blot analysis was performed for Npc1 in various tissues. All three lines demonstrated overexpression of Npc1 in cortex and cerebellum, although the absolute expression levels varied among the three lines. The highest transgene expression was observed for the Tg(Npc1)C3 line, with 12-fold higher expression than wild-type in the cerebellum and 24-fold higher expression in the cortex (Fig. 2A; Table 1). Transgenic lines Tg(Npc1)C2 and Tg(Npc1)B2 also demonstrated overexpression in cortex and cerebellum, although less than was seen in Tg(Npc1)C3 (Fig. 2A). In visceral tissues Npc1 transgene levels examined in liver were less than wild-type expression levels, while those in spleen were close to endogenous levels for Tg(Npc1)B2 and Tg(Npc1)C3 (Fig. 2A, Table 1). Thus, while there was a variation in expression levels between the transgenic lines, there were much higher levels of expression in the CNS as compared with endogenous levels of Npc1 expression (Fig. 2A). Western blot analysis confirmed that NPC1 protein was translated in the cortex and cerebellum of Tg(Npc1)B2 (Fig. 2B), Tg(Npc1)C2 and Tg(Npc1)C3 mice (data not shown).
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Histological analysis of brain, liver and spleen was performed on npc1-/-, Tg(Npc1) animals from the three lines to evaluate the degree of tissue specificity of phenotypic correction for the transgenic lines. In non-transgenic npc1-/- mice at 10 weeks, ataxia was readily apparent upon physical examination of the animals and neurodegeneration was evident upon histological analysis of brain sections (Fig. 3). These mice exhibited necrosis and a near complete loss of Purkinje cells in the cerebellum and axonal spheroids were present in the brain stem. Storage neurons (neuronal cytoplasmic vacuolation) were also present in several areas of the brain (data not shown). Consistent with the rescue of viability and ataxia for the npc1-/-, Tg(Npc1) mice, there was also a rescue of the cellular neurodegeneration. For each of the three Tg(Npc1) lines Purkinje cells were present, no axonal spheroids were observed (Fig. 3) and few neurons with storage were observed in one of the Tg(Npc1) lines (data not shown).
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Brains of the npc1-/-, Tg(Npc1) mice were analysed by immunocytochemistry for evidence of GM2 and GM3 ganglioside accumulation, since extensive ganglioside storage is one hallmark of NPC disease (16). Brain tissue from all three transgenic lines revealed greatly reduced GM2-IR (immunoreactivity) with evidence of only scattered positively stained neurons and/or microglia, with the latter being most conspicuous (Fig. 4). Double-labeling with MAC1 confirmed that most GM2-IR cells in npc1-/-, Tg(Npc1) mice were microglia (Fig. 4) as compared with neurons (data not shown). GM3-immunostaining revealed a similar pattern but less prominent accumulation of this ganglioside in all three transgenic lines (data not shown). The type of labeling observed was similar to that seen in npc1-/- mice, although far fewer cells were affected. Normal mice at this age exhibit little or no evidence of these gangliosides.
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While all three lines of transgenic mice were able to complement the wasting, ataxia and lethality, comparison of GM2-IR between the different lines revealed some distinctive features. Examination of sections from the Tg(Npc1)B2 line revealed widespread GM2-IR in microglial cells scattered throughout the cerebral cortex, hippocampus and nearby subcortical areas, brain stem and cerebellum. Significant neuronal labeling with GM2 and GM3 antibodies was not evident in any part of the CNS in the Tg(Npc1)B2 transgenic line, indicating a lack of ganglioside storage in these cells (Fig. 4C). Brain sections from the Tg(Npc1)C2 line, in addition to microglial labeling similar to Tg(Npc1)B2 animals, also exhibited scattered GM2 and GM3-IR neurons in cerebral cortex, hippocampus and other subcortical areas. The scattered neurons in the deep cerebellar nuclei in the Tg(Npc1)C2 line were particularly positive for GM2 accumulation (Fig. 4D), although neurons in the cerebellar cortex were essentially unstained. The Tg(Npc1)C3 line showed the most GM2 and GM3 labeling, with this occurring both in microglia and in cortical neurons (Fig. 4A). In the cerebellar cortex and deep nuclei there were only scattered GM2-IR cells, mostly glia and their distribution in the granule cell layer was greater than that seen in the other animals. Since npc1-/- mice are known to exhibit severe cerebellar pathology (16,21), parvalbumin and calbindin antibody labeling was used to assess the cellular integrity of this brain region. In all cases, the cerebellar cortex appeared normal, with no evidence for loss of Purkinje cells (data not shown). Consistent with this, Purkinje cells lacked GM2-IR in each of the three transgenic lines described, whereas these neurons in npc1-/- mice typically exhibit significant GM2-IR (16).
In npc1-/- animals, hepatomegaly and splenomegaly are present in tissues at birth (data not shown). Liver and spleen of affected animals show extensive cytoplasmic accumulation of lipid (histiocytosis). For the spleen, this histiocytosis occurred in both the red and white pulp. We evaluated the degree of histiocytosis present in transgenic npc1-/-, Tg(Npc1) animals, in comparison to npc1+/+ and non-transgenic npc1-/-, animals (Fig. 5). For npc1-/-, Tg(Npc1)B2 and npc1-/-Tg(Npc1)C2 mice, liver tissue histology was normal. However, there was minor histiocytosis in the splenic red pulp in these two lines. These results indicate that there was partial rescue of the phenotype in visceral tissues. This rescue may reflect non-restricted transgene expression within these tissues for these two lines (Table 1). For the npc1-/-, Tg(Npc1)C3 line, little rescue of the visceral tissue phenotype occurred. The npc1-/-, Tg(Npc1)C3 animals continued to have moderate hepatic histiocytosis, in addition to moderate splenic histiocytosis located in both the red and white pulp. Further analysis is needed to examine the correlation between transgene expression and histiocytosis on a cellular basis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present study we used npc1-/- mice mated to three prion promoter-driven transgenic lines to demonstrate that rescue of viability and neurological function can be achieved independently from visceral correction of disease. Previously an attempt to correct the clinical course of the disease was performed by mating npc1-/- mice to ß-1-4-GalNAc transerfase null mice (17). The resulting npc1-/-, GalNAcT-/- mice appeared unaltered in terms of clinical progression of the disease, visceral pathology and Purkinje cell loss, although demonstrating a reduced amount of GM2 accumulation in cells relative to npc1-/- mice. The authors concluded from this that the accumulation of GM2 and higher gangliosides are not the primary cause of NPC neurodegeneration and this led to the speculation that the visceral component of the disorder might contribute to the neurological decline observed.
Our study resulted in three lines of transgenic mice that complement different defects that present in npc1-/- mice and are summarized in Table 2. Each of the lines has been able to correct the clinical assayable neurological phenotypes, ataxia and wasting, which are present in the npc1-/- mouse model. Histological analysis indicated that each of the cerebella looked normal with no evidence of Purkinje cell loss or ganglioside accumulation in Purkinje cells. In terms of the visceral phenotype correction, only two of the three lines Tg(Npc1)B2 and Tg(Npc1)C2, displayed significantly corrected histological morphology of liver and spleen when compared with npc1-/- littermates. The third line, Tg(Npc1)C3, continued to have moderate histiocytosis in both the liver and spleen, but remained viable throughout the 18 month observation period, without ataxia or wasting. The Tg(Npc1)C3 transgenic line thus indicates that the observed visceral defects are not making a significant contribution to CNS dysfunction. We suggest that the visceral tissue involvement is not required to generate the neurological phenotype and that correction of the Purkinje cell metabolic defect and rescue of Purkinje cells from dying are fundamental requisites for clinical correction of the wasting, ataxia and early death as seen in NPC.
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Additional supporting evidence that the effects of the visceral phenotype are not contributing to neurological progression comes from studies involving manipulation of cholesterol levels in NPC individuals. Attempts at lowering somatic cholesterol have not had a significant effect on neurological deterioration. Treatment of NPC patients with cholesterol-lowering agents has been shown to lower plasma liver cholesterol levels, but this was without significant clinical effect (22). Dimethyl sulfoxide (DMSO) has been claimed to elicit clinical improvement in one patient (23), while cholestyramine and lovastatin were reported to improve the MRI assessment of lipid accumulation after 3 months in another case (24). It has also been found that nifedipine and probucol treatments, which effectively lower liver cholesterol, do not affect progression of the CNS disease in npc1-/- mice, while absence of the low density lipoprotein receptor (LDLR) had only a very minor effect in delaying the onset of neurological symptoms (25).
It was of great interest that the two transgenic lines tested were fertile, as this correction of fertility allows for direct breeding of npc1-/- animals from npc1-/-, Tg(Npc1) mice. Although npc1-/- mutant mice have always been described as sterile (13,26), there have been few studies of the mechanisms involved. Male npc1-/- mice have low serum testosterone levels, hypothesized to result from a low secretion of testosterone from Leydig cells, as electron microscopy of the Leydig cells showed an extensive accumulation of inclusion bodies and a distorted ER (27). It is important to note that the sterility defect observed in npc1-/- animals may be near a threshold and can be influenced by genetic background effects. This has been demonstrated in the observations that male mice on the F2 129/0la, BALB/cJ background were sometimes fertile and introduction of the mdr1a-/- state by breeding in the knockout (28) corrected female sterility (29).
The expression patterns we found for NPC1 reflect the generally expected pattern from the prion promoter with highest expression of the transgene found in CNS tissues. The original tissue localization of prion protein expression detected by northern analyses showed high levels of expression in brain and lung with undetectable expression in spleen (30). Further studies documented both neuronal (31,32) and glial cell expression (33) in the brain. Extraneuronal sites in the embryo included the tooth primordium and metanephic cap derivatives of the kidney. These general patterns of expression were mostly replicated when the bovine prion protein promoter was used to drive green fluorescent protein expression in transgenic mice (34). Additional areas of expression detected by this sensitive method included 10–15% of T and B lymphocytes, keratinocytes and endothelial cells of the intestine while it is notable that the lung, pancreas, liver and smooth and skeletal muscle were negative (34).
All three transgenic lines showed much higher levels of expression of Npc1 in total CNS tissues than were found in normal mice. These results show that excess Npc1 expression in the CNS is not incompatible with viability, an important observation for potential gene therapy. In some gene therapies, excess gene product can be harmful and create a narrow ‘window’ of dosage which can be tolerated. Analysis of our three transgenic lines indicated that, although the transgene was able to rescue viability of the animals, there was not complete rescue of ganglioside accumulation in a population of CNS neuronal and microgial cells. We used GM2 and GM3 immunostaining as a marker to evaluate the degree to which ganglioside accumulation, a characteristic of NPC (16), was corrected. Among the three lines there was variation in the degree of neuronal and microglial involvement. The Tg(Npc1)C3 line had the most GM2 and GM3 accumulation in the microglial cell population, although this line exhibited the highest level of Npc1 mRNA expression. Whereas Tg(Npc1)B2 had the most correction, the least pathological variation and the lowest Npc1 mRNA expression suggesting a dosage window for GM2 and GM3 accumulation (Table 2). However, these results do not answer the question of whether both neurons and glia, or only one of the two, needed to be corrected since the prion promoter leads to expression in both. Further analysis is needed to determine the correlation between GM2 and GM3 accumulation relative to Npc1 transgene expression in order to more fully characterize the cells types in which the transgene is expressed and this will be the focus of future studies.
In the present study, we have used a transgene to express NPC1 predominantly in the CNS as a method of assessing the relative contribution of visceral versus CNS storage to the pathophysiology of the disease. Thus, we have generated mice that continue to have hepatosplenomegaly, while the neurodegenerative aspects of the disorder have been corrected. Thus the neurodegeneration that occurs in affected individuals is a cell-intrinsic event and not due to the accumulation of toxic by-products relating to the visceral cellular defects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tg(Npc1) construct
A transgenic expression construct was generated with the 3.5 kb prion promoter used to drive expression of the murine Npc1 cDNA. The construct was generated by ligation of the 4 kb Npc1 cDNA KpnI/DraI fragment into the KpnI/EcoRV cut vector PrPmtAde. The resulting 8 kb transgenic construct was linearized with SmaI/MluI and injected into fertilized one-cell oocytes collected from FVB/N females. Microinjected oocytes were then implanted into CByB6F1/J pseudopregnant females. Tail biopsies were taken from the pups for screening of founders. Founder mice were identified using Npc1 probe and PCR genotyping using primers PolyA prpR-GGGGAGGGGCAAACAACAGA and NPC13F-GAGCCACTCATGGACTAATA.
Growth evaluation
Npc1N were maintained as an inbred colony at the National Institute of Health. The Npc1N allele has also been referred to as BALB/cStCrlfC3HfNctr and BALB/c npcnih. Each of the three lines of npc1-/-, Tg(Npc1) animals were crossed with npc1+/-, Tg(Npc1) animals. Resulting male progeny were genotyped and weighed once a week from 4 to 9 weeks of age. All npc1-/- mice obtained demonstrated the disorder and were sacrificed between 8 and 10 weeks of age. For all three transgenic lines, transgene positive npc1-/-, Tg(Npc1) mice exhibited no weight loss and were viable up to 18 months.
Neurological evaluation
Age-matched 10-week-old male mice of appropriate genotype were compared. Front paws were marked with red ink and back paws were marked with blue ink. Mice were allowed to walk on paper through a tunnel 24 inches long and 5 inches wide. Prints spanning 15 inches were scanned and stride measured.
Tissue histology and immunostaining
Animals were anesthetized with 0.4 cm3 Avertin and were then perfused for 3 min with 10 U/ml heparin, 1x phosphate-buffered saline (PBS), followed by 7 min perfusion with 4% paraformaldehyde, 1x PBS. Tissues were dissected and fixed in 4% paraformaldehyde, 1x PBS overnight. For histologic analysis, tissues were obtained from 10-week-old mice, paraffin embedded, sectioned and stained with H&E. Mice 10–16 weeks old were analysed by immunostaining of GM2, GM3 gangliosides, MAC1, parvalbumin and calbindin and methods were followed as described previously (16).
Northern blot analysis
Tissues for RNA isolation were obtained from 8-week-old npc1+/+, Tg(Npc1) animals from each of the three transgenic lines. RNA was prepared using Trizol as per manufacturer's instructions (Gibco, Carlsbad, CA, USA) and 12.5 µg of RNA were loaded per lane. Northern blots were performed as described previously using 1 kb EcoR1 fragment as a probe (6). Npc1 signal was visualized using Typhoon scanner (Molecular Dynamics, Piscataway, NJ, USA). Bands were quantified using ImageQuant software using local background correction (Molecular Dynamics, Piscataway, NJ, USA). Blots were stripped and reprobed with an L-32 RNA labeled probe to provided loading control, as described previously (6).
Western blot analysis
Samples were separated using 7% sodium dodecylsulfate–polyacrylamide gel electrophoresis under reduced conditions and transferred to a nitrocellulose membrane for immunoblot analysis. Immunoblot buffer (0.01 M sodium phosphate, pH 7.4, 0.15 M NaCl, 0.05% Tween 20 and 4% non-fat dry milk) was used for blocking non-specific sites for 2 h. Immunoblots were incubated overnight at 4°C with the primary antibody (to a near-C-terminus peptide) (10) followed by three 10 min rinses. Appropriate peroxidase-conjugated secondary antibodies were incubated for 1 h at room temperature followed by three 10 min rinses. Enhanced chemiluminescence was performed using SuperSignal for Western Blotting (Pierce, Rockford, IL, USA). Protein bands were detected using BioMax MR film (Eastman Kodak, Rochester, NY, USA) and quantitated with a Model GS-700 imaging densitometer (Bio-Rad, Hercules, CA, USA). Background counts were subtracted from each sample. For reprobing immunoblots, antibodies were removed using Immunopure IgG elution buffer (3x30 min at 37°C). Reprobing was performed with mouse monoclonal antibody to B-actin (1 : 10 000; Sigma, St Louis, MO, USA) to better quantitate the amount of protein loaded in each lane.
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ACKNOWLEDGEMENTS |
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We would like to thank Dr Charles Weissman for the prion promoter, Suzanna Gispert for the PrPmtAde construct, Amy Chen for injection of Tg(Npc1) construct, Gene Elliott, Antje Becker, Monica Kiela and Robert McGlenn for technical help and Carole Meyer and Carolyn Henley for secretarial support. This work was supported in part by grants from the Ara Parseghian Medical Research Foundation (W.J.P., S.U.W.).
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FOOTNOTES |
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* To whom correspondence should be addressed at: National Institutes of Health, National Human Genome Research Institute, Genetic Disease Research Branch, 49 Convent Dr., Building 49, Room 4A67, Bethesda, MD 20892, USA. Tel: +1 3014967584; Email: bpavan{at}nhgri.nih.gov
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REFERENCES |
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