Axon-dominant localization of cell-surface cholesterol in cultured hippocampal neurons and its disappearance in Niemann-Pick type C model cells.
ABSTRACT There is growing evidence showing the important role of cholesterol in maintaining neuronal function. In particular, much attention has been paid to the role of the cholesterol-rich microdomains called lipid rafts. However, the cholesterol distribution on neurons is not clear. Here, we investigated localization of cholesterol in cultured rat hippocampal neurons, using filipin and a novel cholesterol-binding reagent BCtheta. In our culture system, BCtheta detects only cell-surface cholesterol, whereas filipin stains both intracellular and cell-surface cholesterol. BCtheta staining appeared visible in a maturation-dependent manner and showed axon-dominant distribution of cell-surface cholesterol in fully matured neurons. A part of this cholesterol on axons was resistant to detergents at 4 degrees C, and thus might be involved in lipid rafts. Interestingly, Niemann-Pick type C model neurons induced by class 2 amphiphiles lost the cell-surface but not the intracellular cholesterol staining. Niemann-Pick type C disease is caused by the disruption of intracellular cholesterol transport and is known to induce neurodegeneration in brains accompanied by formation of neurofibrillary tangles. Our observations suggest the important role of cell-surface cholesterol in maintaining a functional axonal membrane and indicate that the observed defect in axonal surface cholesterol might lead to neurodegeneration.
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ABSTRACT: BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.The Journal of Lipid Research 06/2003; 44(5):1033-41. · 4.39 Impact Factor
Article: Lipid sorting in epithelial cells.Biochemistry 09/1988; 27(17):6197-202. · 3.38 Impact Factor
Article: Role of glia in synapse development.[show abstract] [hide abstract]
ABSTRACT: Recent studies suggest that glial cells regulate certain aspects of synapse development. Neurons can form synapses without glia, but may require glia-derived cholesterol to form numerous and efficient synapses. During synapse maturation, soluble and contact-dependent factors from glia may influence the composition of the postsynaptic density. Finally, synaptic connections appear to require glia to support their structural stability. Given the new evidence, it may be time now to acknowledge glia as a source for synaptogenesis-promoting signals. Scrutinizing the molecular mechanisms underlying this new function of glia and testing its relevance in vivo may help to understand how synapses develop and why they degenerate under pathological conditions.Current Opinion in Neurobiology 11/2002; 12(5):486-90. · 7.34 Impact Factor