Filipin complex-reactive brain lesions: a cautionary tale.

Lau, Adeline A; Trim, Paul J; King, Barbara M; Hassiotis, Sofia; Hung, Ya Hui; Bush, Ashley I; Snel, Marten F; Hemsley, Kim M

Lau, Adeline A; Trim, Paul J; King, Barbara M; Hassiotis, Sofia; Hung, Ya Hui; Bush, Ashley I; Snel, Marten F; Hemsley, Kim M.

Afiliação

Lau AA; Childhood Dementia Research Group, Flinders Health and Medical Research Institute, Flinders University, Bedford Park, South Australia, 5042, Australia.
Trim PJ; Proteomics, Metabolomics and MS-Imaging Core Facility, SAHMRI, Adelaide, South Australia, 5000, Australia.
King BM; Childhood Dementia Research Group, Flinders Health and Medical Research Institute, Flinders University, Bedford Park, South Australia, 5042, Australia.
Hassiotis S; Childhood Dementia Research Group, Flinders Health and Medical Research Institute, Flinders University, Bedford Park, South Australia, 5042, Australia.
Hung YH; Histopathology and Biological Imaging, Future Industries Institute, University of South Australia.
Bush AI; Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, 3052, Australia.
Snel MF; Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, 3052, Australia.
Hemsley KM; Proteomics, Metabolomics and MS-Imaging Core Facility, SAHMRI, Adelaide, South Australia, 5000, Australia.

Neuropathol Appl Neurobiol ; : e12950, 2023 Dec 19.

Article em En | MEDLINE | ID: mdl-38112248

ABSTRACT

ABSTRACT

OBJECTIVE:

Filipin 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.

METHODS:

Brain 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.

RESULTS:

As 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.