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Pitfalls in the detection of cholesterol in Huntington’s disease models
Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington’s disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493072/ https://www.ncbi.nlm.nih.gov/pubmed/23145355 http://dx.doi.org/10.1371/505886e9a1968 |
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author | Marullo, Manuela Valenza, Marta Leoni, Valerio Caccia, Claudio Scarlatti, Chiara De Mario, Agnese Zuccato, Chiara Di Donato, Stefano Carafoli, Ernesto Cattaneo, Elena |
author_facet | Marullo, Manuela Valenza, Marta Leoni, Valerio Caccia, Claudio Scarlatti, Chiara De Mario, Agnese Zuccato, Chiara Di Donato, Stefano Carafoli, Ernesto Cattaneo, Elena |
author_sort | Marullo, Manuela |
collection | PubMed |
description | Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington’s disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner. *Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it |
format | Online Article Text |
id | pubmed-3493072 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-34930722012-11-09 Pitfalls in the detection of cholesterol in Huntington’s disease models Marullo, Manuela Valenza, Marta Leoni, Valerio Caccia, Claudio Scarlatti, Chiara De Mario, Agnese Zuccato, Chiara Di Donato, Stefano Carafoli, Ernesto Cattaneo, Elena PLoS Curr HD Models Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington’s disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner. *Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it Public Library of Science 2012-10-11 /pmc/articles/PMC3493072/ /pubmed/23145355 http://dx.doi.org/10.1371/505886e9a1968 Text en http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | HD Models Marullo, Manuela Valenza, Marta Leoni, Valerio Caccia, Claudio Scarlatti, Chiara De Mario, Agnese Zuccato, Chiara Di Donato, Stefano Carafoli, Ernesto Cattaneo, Elena Pitfalls in the detection of cholesterol in Huntington’s disease models |
title | Pitfalls in the detection of cholesterol in Huntington’s disease models |
title_full | Pitfalls in the detection of cholesterol in Huntington’s disease models |
title_fullStr | Pitfalls in the detection of cholesterol in Huntington’s disease models |
title_full_unstemmed | Pitfalls in the detection of cholesterol in Huntington’s disease models |
title_short | Pitfalls in the detection of cholesterol in Huntington’s disease models |
title_sort | pitfalls in the detection of cholesterol in huntington’s disease models |
topic | HD Models |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493072/ https://www.ncbi.nlm.nih.gov/pubmed/23145355 http://dx.doi.org/10.1371/505886e9a1968 |
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