Filipin complex-reactive brain lesions: A cautionary tale

ObjectiveFilipin complex is an autooxidation-prone fluorescent histochemical stain used in the diagnosis of Niemann-Pick Disease Type C (NP-C), a neurodegenerative lysosomal storage disorder. It is also widely used by researchers examining the distribution and accumulation of unesterified cholesterol in cell and animal models of neurodegenerative diseases including NP-C and Sanfilippo syndrome (mucopolysaccharidosis IIIA; MPS IIIA). Recently, it has been suggested to be useful in studying Alzheimer's and Huntington's disease. Given filipin's susceptibility to photobleaching, we sought to establish a quantitative biochemical method for free cholesterol measurement.MethodsBrain tissue from mice with MPS IIIA was stained with filipin. Total and free cholesterol in brain homogenates was measured using a commercially available kit and a quantitative LC-MS/MS assay was developed. Gangliosides GM1, GM2 and GM3 were also quantified using LC-MS/MS.ResultsAs anticipated, the MPS IIIA mouse brain displayed large numbers of filipin-positive intra-cytoplasmic inclusions, presumptively endo-lysosomes. Challenging the prevailing dogma, however, we found no difference in the amount of free cholesterol in MPS IIIA mouse brain homogenates cf. control tissue, using either the fluorometric kit or LC-MS/MS assay. Filipin has previously been reported to bind to GM1 ganglioside, however, this lipid does not accumulate in MPS IIIA cells/tissues. Using a fluorometric assay, we demonstrate for the first time that filipin cross-reacts with both GM2 and GM3 gangliosides, explaining the filipin-reactive inclusions observed in MPS IIIA brain cells.ConclusionFilipin is not specific for free cholesterol, and positive staining in any setting should be interpreted with caution. Filipin-positive cytoplasmic inclusions are seen in MPS IIIA mouse brain (arrows; A); however, the overall amount of total and unesterified cholesterol does not change in MPS IIIA mouse brain homogenates (B, Amplex Red kit; C, LC-MS/MS). Instead, it is likely that filipin is interacting with GM2 and GM3 gangliosides that have been previously reported to accumulate in MPS IIIA brain, as filipin interacts with each of these gangliosides in addition to GM1 (D, quenching of filipin fluorescence quantified using fluorimetry). Thus, filipin complex staining should be interpreted with extreme caution, as it is not specific for unesterified cholesterol.image