Localization of Niemann–Pick C1 protein in astrocytes: Implications for neuronal degeneration in Niemann– Pick type C disease

Abstract

Niemann-Pick type C disease (NP-C) is an inherited neurovisceral lipid storage disorder characterized by progressive neurodegeneration. Most cases of NP-C result from inactivating mutations of NPC1, a recently identified member of a family of genes encoding membrane-bound proteins containing putative sterol sensing domains. By using a specific antipeptide antibody to human NPC1, we have here investigated the cellular and subcellular localization and regulation of NPC1. By light and electron microscopic immunocytochemistry of monkey brain, NPC1 was expressed predominantly in perisynaptic astrocytic glial processes. At a subcellular level, NPC1 localized to vesicles with the morphological characteristics of lysosomes and to sites near the plasma membrane. Analysis of the temporal and spatial pattern of neurodegeneration in the NP-C mouse, a spontaneous mutant model of human NP-C, by amino-cupric-silver staining, showed that the terminal fields of axons and dendrites are the earliest sites of degeneration that occur well before the appearance of a neurological phenotype. Western blots of cultured human fibroblasts and monkey brain homogenates revealed NPC1 as a 165-kDa protein. NPC1 levels in cultured fibroblasts were unchanged by incubation with low density lipoproteins or oxysterols but were increased 2-to 3-fold by the drugs progesterone and U-18666A, which block cholesterol transport out of lysosomes, and by the lysosomotropic agent NH 4 Cl. These studies show that NPC1 in brain is predominantly a glial protein present in astrocytic processes closely associated with nerve terminals, the earliest site of degeneration in NP-C. Given the vesicular localization of NPC1 and its proposed role in mediating retroendocytic trafficking of cholesterol and other lysosomal cargo, these results suggest that disruption of NPC1-mediated vesicular trafficking in astrocytes may be linked to neuronal degeneration in NP-C.

Secretion of Sterols and the NPC2 Protein from Primary Astrocytes

“…[4][5][6][7][8][9][10][11][12][13][14] C]Cholesterol (specific activity 58 mCi/mmol), [1␣,2␣(n)- 3 H]cholesterol (specific activity 41.0 Ci/mmol), [ 3 H]acetic acid (specific activity 5.10 -9.00 Ci/mmol), Hybond-C membrane, and the ECL reagents were purchased from Amersham Biosciences. The polyclonal anti-glial fibrillary acidic protein antibody was from Dako, and anti-lysobisphosphatidic acid antibody (13) was a gift from Jean Gruenberg (University of Geneva).…”

“…In the NPC1 Ϫ/Ϫ mouse astrocytes are considered to contribute to neurodegeneration (9,10). Interestingly, NPC1 Ϫ/Ϫ astrocytes were shown to have defects in the trafficking of endogenously synthesized cholesterol (11).…”

Cholesterol Movement in Niemann-Pick Type C Cells and in Cells Treated with Amphiphiles

“…The trans-Golgi cisternae in NP-C cells are also enriched in cholesterol (1,6). NPC1, the affected gene in this disease, encodes a polytopic integral membrane glycoprotein that is concentrated in small vesicles bearing lysosomal markers but not much cholesterol (7)(8)(9)(10)(11). It has been speculated that these vesicles facilitate the transport of endocytic cholesterol to the plasma membrane; however, the mechanisms mediating intracellular cholesterol transport are not well understood (12).…”

Intracerebellar Transplantation of Neural Stem Cells into Mice with Neurodegeneration Improves Neuronal Networks with Functional Synaptic Transmission

“…MHP36 were cultured in fibronectin-coated (Sigma-Aldrich, St. Louis, MO, U.S.A.) Flask at 33C with Dulbecco's modified Eagle medium/F12 (DMEM/F12) (Invitrogen, Carlsbad, CA, U.S.A.) supplemented with bovine serum albumin (MP Biomedicals, Aurora, OH), apo transferrin (Sigma-Aldrich) 94 g/ml, putrescine dihydrochloride (Sigma-Aldrich) 15.3 g/ml, insulin (Sigma-Aldrich) 4.7 g/ml, L-thyroxine (SigmaAldrich) 380 ng/ml, tri-iodo-L-thyronine (Sigma-Aldrich) 317 ng/ml, L-glutamine (Invitrogen) 1.9 mM, progesterone (Sigma-Aldrich) 58 ng/ml, sodium selenite (Sigma-Aldrich) 38 ng/ml, heparin sodium salt (Sigma-Aldrich) 9.4 unit/ml, basic fibroblast growth factor (FGF) (Peprotech, Rocky Hill, NJ, U.S.A.) 10 ng/ml, interferon (Peprotech) 1.2 ng/ml. The culture condition was maintained in an undifferentiated state at 33C and 5% CO 2 [19,20]. For transplantation, the cells were liberated from culture flasks using versene (Invitrogen) rather than a trypsin-based technique to preserve protein expression, and were pelleted from Hank's Balanced Salt Solution (HBSS).…”

“…In most neurodegenerative diseases characterized by inflammation and another neurodegenerative disease, Niemann-pick disease type C (NP-C), chronic inflammatory reaction has been considered to be a key process leading to neuronal degeneration [1,4,20]. NP-C is an inherited lysosomal storage disorders resulting from a mutation of the NPC gene, and characterized by progressive ataxia, cerebellar atrophy, psychomotor deterioration, extrapyramidal deficits, and dementia.…”