title: Pitfalls in the detection of cholesterol in Huntington's disease models.
abstract: 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.
authors.affiliation: Centre for Stem Cell Research, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Marullo
authors.forename: Marta
authors.initials: M
authors.lastname: Valenza
authors.forename: Valerio
authors.initials: V
authors.lastname: Leoni
authors.forename: Claudio
authors.initials: C
authors.lastname: Caccia
authors.forename: Chiara
authors.initials: C
authors.lastname: Scarlatti
authors.forename: Agnese
authors.initials: A
authors.lastname: De Mario
authors.forename: Chiara
authors.initials: C
authors.lastname: Zuccato
authors.forename: Stefano
authors.initials: S
authors.lastname: Di Donato
authors.forename: Ernesto
authors.initials: E
authors.lastname: Carafoli
authors.forename: Elena
authors.initials: E
authors.lastname: Cattaneo