Filipin complex-reactive brain lesions: a cautionary tale.

IF 4 2区医学 Q1 CLINICAL NEUROLOGY

Neuropathology and Applied Neurobiology Pub Date : 2023-12-19 DOI:10.1111/nan.12950

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

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引用次数: 0

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.

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菲利宾复合体反应性脑损伤：一个值得警惕的故事。

目的：菲力蛋白复合物是一种易自体氧化的荧光组织化学染色剂，用于诊断神经退行性溶酶体贮积症--尼曼病 C 型（NP-C）。它还被研究人员广泛用于检查未酯化胆固醇在神经退行性疾病（包括 NP-C 和 Sanfilippo 综合征（粘多糖病 IIIA；MPS IIIA））的细胞和动物模型中的分布和积累情况。最近，有人认为它有助于研究阿尔茨海默氏症和亨廷顿氏病。鉴于丝裂蛋白易受光漂白影响，我们试图建立一种定量测量游离胆固醇的生化方法：方法：用丝蛋白对患有 MPS IIIA 的小鼠的脑组织进行染色。使用市售试剂盒测量脑匀浆中的总胆固醇和游离胆固醇，并开发出一种 LC-MS/MS 定量分析方法。还使用 LC-MS/MS 对神经节苷脂 GM1、GM2 和 GM3 进行了定量：正如预期的那样，MPS IIIA 小鼠大脑显示出大量丝裂蛋白阳性的胞浆内包涵体，推测为内溶酶体。然而，我们发现 MPS IIIA 小鼠脑匀浆中游离胆固醇的含量与对照组织相比并无差异，这对流行的教条提出了挑战。以前曾有报道称，菲力平可与 GM1 神经节苷脂结合，但这种脂质不会在 MPS IIIA 细胞/组织中积累。利用荧光测定法，我们首次证明了丝裂蛋白与 GM2 和 GM3 神经节苷脂有交叉反应，从而解释了在 MPS IIIA 脑细胞中观察到的丝裂蛋白反应性包涵体：结论：菲利平对游离胆固醇没有特异性，在任何情况下都应谨慎解释阳性染色。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Neuropathology and Applied Neurobiology 医学-病理学

CiteScore

8.20

自引率

2.00%

发文量

审稿时长

6-12 weeks

期刊介绍： Neuropathology and Applied Neurobiology is an international journal for the publication of original papers, both clinical and experimental, on problems and pathological processes in neuropathology and muscle disease. Established in 1974, this reputable and well respected journal is an international journal sponsored by the British Neuropathological Society, one of the world leading societies for Neuropathology, pioneering research and scientific endeavour with a global membership base. Additionally members of the British Neuropathological Society get 50% off the cost of print colour on acceptance of their article.