A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain
2004; Elsevier BV; Volume: 45; Issue: 3 Linguagem: Inglês
10.1194/jlr.d300032-jlr200
ISSN1539-7262
AutoresPatrick Reid, Naomi Sakashita, Shigeki Sugii, Yoshiko Ohno‐Iwashita, Yukiko Shimada, William F. Hickey, Ta‐Yuan Chang,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoNiemann-Pick type C (NPC) is a neurodegenerative disorder characterized by progressive accumulation of cholesterol, gangliosides, and other lipids in the central nervous system and visceral organs. In the NPC1 mouse model, neurodegeneration and neuronal cell loss occur before postnatal day 21. Whether neuronal cholesterol accumulation occurs in vivo before the first signs of neuronal cell loss has not been demonstrated. In this report, we used the NPC1 mouse model and employed a novel cholesterol binding reagent, BCθ, that enabled us to visualize cellular cholesterol accumulation at a level previously unattainable.The results demonstrate the superiority of BCθ staining over conventional filipin staining in confocal microscopy and highlight several new findings. We show that at postnatal day 9, although only mild signs of neurodegeneration are detectable, significant neuronal cholesterol accumulation has already occurred throughout the NPC1 brain. In addition, although NPC1 Purkinje neurons exhibit a normal morphology at day 9, significant cholesterol accumulation within their extensive dendritic trees has occurred. We also show that in the thalamus and cortex of NPC1 mice, activated glial cells first appear at postnatal day 9 and heavily populate by day 22, suggesting that in NPC1 mice, neuronal cholesterol accumulation precedes neuronal injury and neuronal cell loss. Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by progressive accumulation of cholesterol, gangliosides, and other lipids in the central nervous system and visceral organs. In the NPC1 mouse model, neurodegeneration and neuronal cell loss occur before postnatal day 21. Whether neuronal cholesterol accumulation occurs in vivo before the first signs of neuronal cell loss has not been demonstrated. In this report, we used the NPC1 mouse model and employed a novel cholesterol binding reagent, BCθ, that enabled us to visualize cellular cholesterol accumulation at a level previously unattainable. The results demonstrate the superiority of BCθ staining over conventional filipin staining in confocal microscopy and highlight several new findings. We show that at postnatal day 9, although only mild signs of neurodegeneration are detectable, significant neuronal cholesterol accumulation has already occurred throughout the NPC1 brain. In addition, although NPC1 Purkinje neurons exhibit a normal morphology at day 9, significant cholesterol accumulation within their extensive dendritic trees has occurred. We also show that in the thalamus and cortex of NPC1 mice, activated glial cells first appear at postnatal day 9 and heavily populate by day 22, suggesting that in NPC1 mice, neuronal cholesterol accumulation precedes neuronal injury and neuronal cell loss. Niemann-Pick type C (NPC) disease is a fatal autosomal recessive neurovisceral disorder in humans and in animals, characterized by progressive neurodegeneration in the central nervous system (CNS) and hepatosplenomegaly. The disease can be caused by mutations in one of two genes, NPC1 and NPC2 [as reviewed in ref. (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick disease type C: a lipid trafficking disorder.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3611-3633Google Scholar)]. Mutations in Npc1 account for 95% of all NPC disease cases, whereas mutations in Npc2 account for the remaining 5% (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick disease type C: a lipid trafficking disorder.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3611-3633Google Scholar). At the cellular level, NPC disease is characterized by the accumulation of unesterified cholesterol, sphingomyelin, glycosphingolipids, and other lipids within the endosomal/lysosomal system in various tissues. The Npc1 gene encodes a multi-pass transmembrane protein with a putative sterol-sensing domain (2Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie E.J. Dwyer N.K. Neufeld E.B. Chang T.Y. Liscum L. Strauss III, J.F. Ohno K. Zeigler M. Carmi R. Sokol J. Markie D. O'Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle D.A. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Crossref PubMed Scopus (1212) Google Scholar). It resides within the tubulovesicles associated with the late endosome/lysosome (3Ko D.C. Gordon M.D. Jin J.Y. Scott M.P. Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events.Mol. Biol. Cell. 2002; 12: 601-614Crossref Scopus (211) Google Scholar, 4Zhang M. Dwyer N.K. Love D.C. Cooney A. Comly M. Neufeld E.B. Pentchev P.G. Blanchette-Mackie E.J. Hanover J.A. Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease.Proc. Natl. Acad. Sci. USA. 2001; 98: 4466-4471Crossref PubMed Scopus (119) Google Scholar). In NPC1 mutant cells, the transport of both LDL-derived and endogenously synthesized cholesterol through the endosome/lysosome is partially defective in a cell type-dependent manner (5Wojtanik K.M. Liscum L. 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Niemann-Pick type C mutations cause lipid traffic jam.Traffic. 2000; 1: 218-225Crossref PubMed Scopus (132) Google Scholar). In vitro, the NPC1 protein exhibits a transmembrane molecular pump activity for fatty acids but not for cholesterol (13Davies J.P. Chen F.W. Ioannou Y.A. Transmembrane molecular pump activity of Niemann-Pick C1 protein.Science. 2000; 290: 2295-2298Crossref PubMed Scopus (254) Google Scholar). The Npc2 gene encodes the soluble protein HE1, a lysosomal protein that can be secreted into the growth medium (14Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Crossref PubMed Scopus (698) Google Scholar). NPC2 binds cholesterol with very high affinity and binds fatty acids with lower affinity (15Ko D.C. Binkley J. Sidow A. Scott M.P. The integrity of a cholesterol-binding pocket in Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels.Proc. Natl. Acad. Sci. USA. 2003; 100: 2518-2525Crossref PubMed Scopus (161) Google Scholar). Despite evidence at the in vitro level favoring the view that NPC1 and NPC2 are involved in intracellular cholesterol transport, the direct connection between cholesterol accumulation and neurodegeneration in NPC brains remains under debate. Earlier studies [as reviewed in ref. (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick disease type C: a lipid trafficking disorder.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3611-3633Google Scholar)] reported that glycolipids are elevated in the NPC1 brain, the primary target of this disease, whereas there is no overt increase in cholesterol in the brain in human NPC1 or its animal models (16Xie C. Turley S.D. Pentchev P.G. Dietschy J.M. Cholesterol balance and metabolism in mice with loss of function Niemann-Pick C protein.Am. J. Physiol. 1999; 276: E336-E344PubMed Google Scholar). The neuropathological abnormalities of NPC1 disease resemble those in primary gangliosidoses (i.e., diseases caused by enzyme deficiencies in the glycolipid degradation pathway). The connection between glycolipids and NPC1 is further supported by the work of Vanier (17Vanier M.T. Lipid changes in Niemann-Pick disease type C brain: personal experience and review of the literature.Neurochem. Res. 1999; 24: 481-489Crossref PubMed Scopus (167) Google Scholar), who reported that the total GM2 and GM3 contents in the cerebral cortex of a three-month-old patient were highly elevated as compared with those of age-matched infants. In addition, Zervas, Dobrenis, and Walkley (18Zervas M. Dobrenis K. Walkley S.U. Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations.J. Neuropathol. Exp. Neurol. 2001; 60: 49-64Crossref PubMed Scopus (220) Google Scholar) used a monoclonal antibody against GM2 to perform immunostaining and showed that the GM2 immunoreactivity increased in the cortical pyramidal neurons in animals and humans with NPC1 in a manner similar to that found in primary GM2 gangliosidosis. The same group of investigators then showed that treating NPC1 animals with the drug N-butyldeoxynojirimycin, an inhibitor of the enzyme glucosylceramide synthase, a key enzyme in the early glycosphingolipid biosynthesis pathway, decreased the ganglioside accumulation and the accompanying neuropathological changes in their brains (19Zervas M. Somers K.L. Thrall M.A. Walkley S.U. Critical role of glycosphingolipids in Niemann-Pick disease type C.Curr. Biol. 2001; 11: 1283-1287Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). These and other studies suggested that NPC1 might be considered a glycolipid storage disease rather than a cholesterol storage disease. However, recent evidence for cholesterol accumulation in the NPC1 brain has been described. In NPC1 mice, Zervas, Dobrenis, and Walkley (18Zervas M. Dobrenis K. Walkley S.U. Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations.J. Neuropathol. Exp. Neurol. 2001; 60: 49-64Crossref PubMed Scopus (220) Google Scholar) reported cholesterol accumulation in various neurons, in addition to GM2 accumulation. The mice examined by these investigators were at 9.5 weeks of age, and were near the end of their life span (which averages between 10 to 11 weeks) (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick disease type C: a lipid trafficking disorder.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3611-3633Google Scholar). In a separate study, Treiber-Held et al. (20Treiber-Held S. Distl R. Meske V. Albert F. Ohm T.G. Spatial and temporal distribution of intracellular free cholesterol in brains of a Niemann-pick type C mouse model showing hyperphosphorylated tau protein. Implications for Alzherimer's disease.J. Pathol. 2003; 200: 95-103Crossref PubMed Scopus (47) Google Scholar) examined cholesterol accumulation in NPC1 mice between 3 and 10 weeks of age. They reported significant cholesterol accumulation in neurons of the cortex, the CA1 region of the hippocampus, and the cerebellar cortex as early as 3 weeks of age (20Treiber-Held S. Distl R. Meske V. Albert F. Ohm T.G. Spatial and temporal distribution of intracellular free cholesterol in brains of a Niemann-pick type C mouse model showing hyperphosphorylated tau protein. Implications for Alzherimer's disease.J. Pathol. 2003; 200: 95-103Crossref PubMed Scopus (47) Google Scholar). In addition, they observed only mild cholesterol accumulation in the CA3 region of the hippocampus, the dentate gyrus, and the thalamus. However, in the NPC1 mouse, neurodegeneration and low levels of neuronal cell loss have been reported to occur before postnatal day 21, with the earliest signs of neurodegeneration reported at postnatal day 9 (21Ong W-Y. Kumar U. Switzer R.C. Sidhu A. Suresh G. Hu C-Y. Patel S.C. Neurodegeneration in Niemann-Pick type C disease mice.Exp. Brain Res. 2001; 141: 218-231Crossref PubMed Scopus (97) Google Scholar, 22German D.C. Liang C-L. Song T. Yazdani U. Xie C. Dietschy J.M. Neurodegeneration in the Niemann-Pick C mouse: glial involvment.Neuroscience. 2002; 109: 437-450Crossref PubMed Scopus (154) Google Scholar). In addition, by day 21 there is already a 13% loss of Purkinje neurons from the cerebellum, a hallmark of NPC disease, which progresses to a 98% loss by the end of the life span (22German D.C. Liang C-L. Song T. Yazdani U. Xie C. Dietschy J.M. Neurodegeneration in the Niemann-Pick C mouse: glial involvment.Neuroscience. 2002; 109: 437-450Crossref PubMed Scopus (154) Google Scholar). Whether significant cholesterol accumulation occurs in vivo before the first signs of neuronal cell loss (pre-postnatal day 21) and neurodegeneration has not been demonstrated. To visualize cholesterol accumulation in cells, various investigators invariably use filipin staining as the standard method [as reviewed in ref. (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Niemann-Pick disease type C: a lipid trafficking disorder.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3611-3633Google Scholar)]. Filipin binds to cholesterol with high affinity; however, it exhibits rapid photobleaching and only modest natural fluorescence under UV excitation, making its detection under confocal microscopy difficult. Recently, a novel method for detecting cholesterol-rich domains utilizing the cholesterol binding agent BCθ was developed. BCθ originates from a protein toxin called theta-toxin produced by Clostridium perfringens. For detection purposes, the toxin has been modified by proteolysis and then biotinylated. By employing avidin-conjugated fluorescent dyes, BCθ bound to cellular cholesterol can be visualized under fluorescence microscopy (23Iwamoto M. Morita I. Fukuda M. Murota S. Ando S. Ohno-Iwashita Y. A biotinylated perfringolysin O derivative: a new probe for detection of cell surface cholesterol.Biochim. Biophys. Acta. 1997; 1327: 222-230Crossref PubMed Scopus (62) Google Scholar). In unfixed cells or cells fixed with 1% or 2% paraformaldehyde, BCθ cannot enter the cell interior and binds mainly to cholesterol-rich domain(s) at the cell surface (24Waheed A.A. Shimada Y. Heijnen H.F. Nakamura M. Inomata M. Hayashi M. Iwashita S. Slot J.W. Ohno-Iwashita Y. Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts).Proc. Natl. Acad. Sci. USA. 2001; 98: 4926-4931Crossref PubMed Scopus (200) Google Scholar, 25Sugii S. Reid P.C. Ohgami N. Shimada Y. Maue R.A. Ninomiya H. Ohno-Iwashita Y. Chang T.Y. Biotinylated theta toxin derivative as a probe to examine intracellular cholesterol domains in normal and Niemann-Pick type C1 cells.J. Lipid Res. 2003; 44: 1033-1041Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In contrast, when cells are fixed with 4% paraformaldehyde, a significant portion of the cholesterol binding sites at the plasma membrane (PM) are inactivated, presumably because of extensive cross-linking of membrane proteins (26Mobius W. Ohno-Iwashita Y. van Donselaar E.G. Oorschot V.M. Shimada Y. Fujimoto T. Heijnen H.F. Geuze H.J. Slot J.W. Immunoelectron microscopic localization of cholesterol using biotinylated and non-cytolytic perfringolysin O.J. Histochem. Cytochem. 2002; 50: 43-55Crossref PubMed Scopus (226) Google Scholar). Concurrently, the cells become leaky to various macromolecules, allowing BCθ to enter the cell interior. At the cell interior, BCθ was shown to stain mainly cholesterol-rich domains; the complexes formed can be visualized under fluorescence microscopy in a manner far superior to that of filipin (25Sugii S. Reid P.C. Ohgami N. Shimada Y. Maue R.A. Ninomiya H. Ohno-Iwashita Y. Chang T.Y. Biotinylated theta toxin derivative as a probe to examine intracellular cholesterol domains in normal and Niemann-Pick type C1 cells.J. Lipid Res. 2003; 44: 1033-1041Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In the current study, we employ this new method to monitor cholesterol accumulation in the brains of NPC1 mice from the first signs of neurodegeneration at postnatal day 9 to a stage of low levels of neuronal cell loss at postnatal day 22. The animal studies were prereviewed and approved by the Institutional Animal Use and Care Committee at Dartmouth College, Hanover, New Hampshire: Protocol #11601. The BALB/c NPC1NIH mice were kindly donated by Peter G. Pentchev at the National Institutes of Health. Mice were bred as NPC+/− heterozygotes. Litters were genotyped via tail snip DNA by a previously described PCR method (27Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Crossref PubMed Scopus (697) Google Scholar). Preliminary studies revealed no detectable abnormal phenotypes in the heterozygous animals (NPC+/−), consistent with previous reports. Homozygous NPC (NPC−/−) mice and their age-matched normal siblings (NPC+/+) were examined at 9, 11, 15, and 22 days of age, with n = 4 animals (2 NPC−/− and 2 NPC+/+) per age point. At each time period, mice were anesthetized with ether and perfused through their hearts with Dulbecco's phosphate buffered saline without calcium and magnesium followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were excised, and large coronal sections were taken with a razor blade through the cerebellum and the underlying brain stem, and through the cortex and the underlying basal structures. The large tissue sections were fixed in 4% paraformaldehyde for 30 min, washed multiple times in 0.2 M phosphate buffer, and cryoprotected in 30% sucrose at 4°C overnight. For each animal, coronal brain sections from the cerebellum/brainstem and the cortex/basal structures were embedded side by side; 5 μm thin sections were cut on a cryostat and placed on precleaned glass slides. Section slides were blocked with 1% fetal bovine serum (FBS) in 0.5 M Tris (pH 7.6), or with PBS containing 1% BSA and the nonspecific mouse IgM antibodies (for GM1 and GM2 slides only), then stained for histochemical analysis as described below. Section slides described above were incubated overnight at 4°C on humidified trays with various primary staining reagents diluted in 0.5 M Tris containing 1% FBS, or in PBS containing 1% BSA (for GM1 and GM2 staining). BCθ was prepared as previously described (24Waheed A.A. Shimada Y. Heijnen H.F. Nakamura M. Inomata M. Hayashi M. Iwashita S. Slot J.W. Ohno-Iwashita Y. Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts).Proc. Natl. Acad. Sci. USA. 2001; 98: 4926-4931Crossref PubMed Scopus (200) Google Scholar) and used at 15 μg/ml. Anti-GM1 biotin-conjugated mouse monoclonal class IgM was from Seikagaku Co., Japan, and was used at 1:100 (v/v). Anti-GM2 mouse monoclonal class IgM, described by Taniguchi et al. (28Taniguchi M. Shinoda Y. Ninomiya H. Vanier M.T. Ohno K. Sites and temporal changes of gangliosides GM1/GM2 storage in the Niemann-Pick disease type C mouse brain.Brain Dev. 2001; 23: 414-421Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), was generously provided by Dr. T. Tai of the Tokyo Metropolitan Institute of Gerontology. For GM1 and GM2 staining, sections were permeabilized with 0.5% Triton X-100 as described previously (28Taniguchi M. Shinoda Y. Ninomiya H. Vanier M.T. Ohno K. Sites and temporal changes of gangliosides GM1/GM2 storage in the Niemann-Pick disease type C mouse brain.Brain Dev. 2001; 23: 414-421Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Filipin (125 μg/ml) staining was performed under light-protected conditions for 2 h at room temperature, as previously described (25Sugii S. Reid P.C. Ohgami N. Shimada Y. Maue R.A. Ninomiya H. Ohno-Iwashita Y. Chang T.Y. Biotinylated theta toxin derivative as a probe to examine intracellular cholesterol domains in normal and Niemann-Pick type C1 cells.J. Lipid Res. 2003; 44: 1033-1041Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). After incubation with primary reagents, slides were washed with 0.5 M Tris containing no FBS and counterstained with NeurotraceTM fluorescent Nissl Stain (N-21480) 500/525 green (Molecular Probes, Eugene, OR) according to the manufacturer's literature. Anti-Calbindin-D-28K (EG-20) rabbit polyclonal antibodies (Sigma) were used to stain Purkinje neurons and their dendritic architecture. Anti-glial acidic fibrillary protein (GFAP) rabbit polyclonal antibodies (Sigma) were used to stain reactive astrocytes. Anti-F4/80 rat monoclonal antibodies (Accurate Chemical and Scientific Corp.) were used to stain macrophages. Bound BCθ, GM1, GM2, GFAP, or Calbindin antibodies were detected with the following secondary reagents: streptavidin-Alexa 488 or -Alexa 568 (at 1:1,000, v/v), goat anti-mouse IgM-Alexa 647 (at 1:150, v/v), horse/goat anti-rabbit-Alexa 488 (at 1:150, v/v), -Alexa 568 (at 1:150, v/v), or Cy 5 (at 1:150, v/v), and donkey anti-rat-Alexa 568 (at 1:150, v/v). All secondary reagents were from Molecular Probes. Slides were treated with Prolong anti-fade from Molecular Probes. Images were collected with a confocal microscope (Bio-Rad MRC-1024) and constructed with LaserSharp software. Images of filipin-stained sections were collected on a Leica TCS-SP confocal microscope equipped with an argon laser for UV excitations, and constructed with Leica Confocal Software. Multiple coronal brain sections from postnatal day 9, 11, 15, and 22 NPC and wild-type (WT) mice were stained with BCθ and Neurotrace. Sections representing cortex, thalamus, hypothalamus, hippocampus, dentate gyrus, and cerebellum were examined under a 10× objective using a Bio-Rad MRC1024 confocal microscope. For each section, six image scans, encompassing all regions of the brain, were taken, and the total image BCθ fluorescent intensity was determined by LaserSharp software. The Alexa dye conjugates used are very photo-stable under confocal microscopy, and no reduction in signal was observed during analysis. Sections from WT mice exhibited low levels of BCθ-positive signals, representing background staining of synaptosomal membranes and myelin-associated cholesterol. Therefore, to serve as a baseline, we adjusted the IRIS and GAIN features of the MRC1024 confocal microscope so that WT day 9 brain sections exhibited mean fluorescent intensities of 1,000 arbitrary units. Once these parameters were set, sections from WT day 11, 15, and 22 and NPC day 9, 11, 15, and 22 mice were scanned and the intensity values measured. To ensure that the measured intensity values were proportional to the signal intensity, instrument settings were chosen such that the signals recorded from the brightest samples prepared from the NPC1 mice did not go beyond the full-scale value. To confirm these findings, in a second experiment, a second set of brains from NPC and WT day 9, 11, 15, and 22 mice was examined in the same manner. Neurotrace staining was used to aid in the identification of regions and as an internal standard to determine differences in staining intensities in NPC and WT sections. Measurements of neurotrace fluorescent intensities showed <15% difference in values, indicating that the differences were specific to BCθ staining. Attempts to perform a similar analysis with filipin proved impossible because of the high background associated with filipin staining at these ages and its rapid bleaching time in confocal microscopy. To compare the ability of BCθ and filipin to detect cholesterol accumulation in vivo, normal (WT) and NPC1 (NPC) mouse brains, taken from postnatal day 9, 15, and 22 genotype-confirmed animals were processed for histochemistry and stained with BCθ and with filipin in parallel. A fluorescent Nissl stain, Neurotrace™ (Molecular Probes), which stains the extensive rough endoplasmic reticulum in neurons, was used as an identifying marker for neurons. As early as postnatal day 9, positive BCθ staining (Fig. 1, arrows), indicative of cholesterol accumulation, was observed in neurons of all regions of the NPC brain, including the cerebellum, cortex, thalamus, granule layer of the dentate gyrus, and the large pyramidal neurons of Ammon's horn in the hippocampus (regions CA1–CA3). This staining was variable from region to region, but in general, the extent of cholesterol accumulation progressed from day 9 to day 22. In WT mouse brains, cholesterol accumulation was undetectable in these regions. In comparison, weak sporadic filipin staining (Fig. 2)in neurons was present in postnatal day 9 NPC brains, with increased signals observed in NPC brains at postnatal day 22, although both NPC and WT brains exhibited high background (Fig. 2).Fig. 2Detection of neuronal cholesterol accumulation by filipin staining. Coronal brain sections (5 μm) taken from sibling WT (NPC1+/+) and NPC (NPC1−/−) mice perfuse-fixed with 4% paraformaldehyde were stained with filipin (blue) and neurotrace (green) and examined by confocal microscopy. Filipin (blue) stains intracellular cholesterol-rich domain(s), indicated by arrows; neurotrace (green), a fluorescent Nissl stain, stains neuronal perikarya, indicated by asterisks. Images shown are high-magnification images obtained from WT and NPC brains at postnatal days 9 and 22, and are representative of a large number of photographs obtained by confocal microscopy from multiple day 9, 11, 15, and 22 WT and NPC mice brains. Abbreviations used: CBL, cerebellum; CTX, cortex; TH, thalamus; HC, hippocampus CA3 region; DG, dentate gyrus. Scale bar is 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In NPC cerebellum, BCθ staining detected low levels of cholesterol accumulation within Purkinje cells (Fig. 1, arrows; CBL indicated by the asterisk) and small granule neurons (Fig. 1; CBL, indicated by the plus sign) of the granule layer as early as postnatal day 9. Cholesterol accumulation in both Purkinje and granule neurons increased from day 9 to day 22 (Fig. 1, CBL). In WT mouse brain, BCθ did not detect any cholesterol accumulation between day 9 and day 22 within the cerebellum (Fig. 1, CBL). In contrast, cholesterol accumulation could not be detected by filipin in postnatal day 9 NPC cerebellum (Fig. 2, CBL). By day 22, filipin staining could detect cholesterol accumulation within Purkinje neurons (Fig. 2, arrows; CBL, asterisk), but not in granule neurons. At day 9, based on morphological appearance, NPC Purkinje cells exhibited no obvious abnormalities, but by day 22, multiple alterations in cell morphology, such as dendritic alterations and axonal swelling, became visually apparent. In addition, there was evidence of missing Purkinje cells when NPC cerebellar sections were stained with Calbindin, a Purkinje cell marker protein, suggesting initial Purkinje cell loss. At this stage, neuronal cell loss in the granular layer was not obvious. In NPC cortex (Fig. 1, CTX), at day 9, significant cholesterol accumulation was observed in neurons of all layers. Accumulation occurred predominantly in the neuronal perikarya (asterisk) as well as the axon hillock region (arrow). Cholesterol accumulations were barely detectable by filipin staining at postnatal day 9, and were difficult to discern from background staining (Fig. 2, CTX). Filipin staining at day 22 showed cholesterol accumulations in the axon hillock region of most neurons (Fig. 2, CTX, indicated by arrows), similar to BCθ staining
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