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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...

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Autores principales: Marullo, Manuela, Valenza, Marta, Leoni, Valerio, Caccia, Claudio, Scarlatti, Chiara, De Mario, Agnese, Zuccato, Chiara, Di Donato, Stefano, Carafoli, Ernesto, Cattaneo, Elena
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
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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
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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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