Filipin complex-reactive brain lesions: A cautionary tale

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

doi:10.1111/nan.12950

Filipin complex-reactive brain lesions: A cautionary tale

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

Research output: Contribution to journal › Article › peer-review

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.

Conclusion

Filipin is not specific for free cholesterol, and positive staining in any setting should be interpreted with caution.

Original language	English
Article number	e12950
Number of pages	8
Journal	Neuropathology and Applied Neurobiology
Volume	50
Issue number	1
DOIs	https://doi.org/10.1111/nan.12950
Publication status	Published - Feb 2024

Keywords

Niemann-Pick C
cholesterol
filipin complex
ganglioside
lysosomal
mouse
mucopolysaccharidosis

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@article{2e831767e6974603ac7bfeec94c4959b,

title = "Filipin complex-reactive brain lesions: A cautionary tale",

abstract = "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.",

keywords = "Niemann-Pick C, cholesterol, filipin complex, ganglioside, lysosomal, mouse, mucopolysaccharidosis",

author = "Lau, {Adeline A.} and Trim, {Paul J.} and King, {Barbara M.} and Sofia Hassiotis and Hung, {Ya Hui} and Bush, {Ashley I.} and Snel, {Marten F.} and Hemsley, {Kim M.}",

year = "2024",

month = feb,

doi = "10.1111/nan.12950",

language = "English",

volume = "50",

journal = "Neuropathology and Applied Neurobiology",

issn = "0305-1846",

publisher = "BLACKWELL PUBLISHERS INC",

number = "1",

}

TY - JOUR

T1 - Filipin complex-reactive brain lesions

T2 - A cautionary tale

AU - Lau, Adeline A.

AU - Trim, Paul J.

AU - King, Barbara M.

AU - Hassiotis, Sofia

AU - Hung, Ya Hui

AU - Bush, Ashley I.

AU - Snel, Marten F.

AU - Hemsley, Kim M.

PY - 2024/2

Y1 - 2024/2

N2 - 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.

AB - 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.

KW - Niemann-Pick C

KW - cholesterol

KW - filipin complex

KW - ganglioside

KW - lysosomal

KW - mouse

KW - mucopolysaccharidosis

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UR - http://purl.org/au-research/grants/nhmrc/565074

U2 - 10.1111/nan.12950

DO - 10.1111/nan.12950

M3 - Article

C2 - 38112248

AN - SCOPUS:85182661155

SN - 0305-1846

VL - 50

JO - Neuropathology and Applied Neurobiology

JF - Neuropathology and Applied Neurobiology

IS - 1

M1 - e12950

ER -

Filipin complex-reactive brain lesions: A cautionary tale

Abstract

Keywords

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