Changes in brain oxysterols at different stages of Alzheimer's disease: Their involvement in neuroinflammation

Gabriella Testa, Erica Staurenghi, Chiara Zerbinati, Simona Gargiulo, Luigi Iuliano, Giorgio Giaccone, Fausto Fantò, Giuseppe Poli, Gabriella Leonarduzzi, Paola Gamba, Gabriella Testa, Erica Staurenghi, Chiara Zerbinati, Simona Gargiulo, Luigi Iuliano, Giorgio Giaccone, Fausto Fantò, Giuseppe Poli, Gabriella Leonarduzzi, Paola Gamba

Abstract

Alzheimer's disease (AD) is a gradually debilitating disease that leads to dementia. The molecular mechanisms underlying AD are still not clear, and at present no reliable biomarkers are available for the early diagnosis. In the last several years, together with oxidative stress and neuroinflammation, altered cholesterol metabolism in the brain has become increasingly implicated in AD progression. A significant body of evidence indicates that oxidized cholesterol, in the form of oxysterols, is one of the main triggers of AD. The oxysterols potentially most closely involved in the pathogenesis of AD are 24-hydroxycholesterol and 27-hydroxycholesterol, respectively deriving from cholesterol oxidation by the enzymes CYP46A1 and CYP27A1. However, the possible involvement of oxysterols resulting from cholesterol autooxidation, including 7-ketocholesterol and 7β-hydroxycholesterol, is now emerging. In a systematic analysis of oxysterols in post-mortem human AD brains, classified by the Braak staging system of neurofibrillary pathology, alongside the two oxysterols of enzymatic origin, a variety of oxysterols deriving from cholesterol autoxidation were identified; these included 7-ketocholesterol, 7α-hydroxycholesterol, 4β-hydroxycholesterol, 5α,6α-epoxycholesterol, and 5β,6β-epoxycholesterol. Their levels were quantified and compared across the disease stages. Some inflammatory mediators, and the proteolytic enzyme matrix metalloprotease-9, were also found to be enhanced in the brains, depending on disease progression. This highlights the pathogenic association between the trends of inflammatory molecules and oxysterol levels during the evolution of AD. Conversely, sirtuin 1, an enzyme that regulates several pathways involved in the anti-inflammatory response, was reduced markedly with the progression of AD, supporting the hypothesis that the loss of sirtuin 1 might play a key role in AD. Taken together, these results strongly support the association between changes in oxysterol levels and AD progression.

Keywords: Alzheimer's disease; Cholesterol metabolism; Inflammation; Oxysterols; Sirtuin-1.

Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1
Fig. 1
Distribution of neurofibrillary changes in AD brain specimens. Immunohistochemistry with monoclonal antibody AT8 to p-tau (immunoreactivity corresponds to brown reaction product) revealed severe involvement of all regions of the cerebral cortex by neurofibrillary changes (C, Braak stage VI), that can spare the primary motor and sensory areas (B, Braak stage IV) or involve selectively the mesial temporal areas (A, Braak stage II).
Fig. 2
Fig. 2
Quantification of oxysterols present in autopsy samples of frontal and occipital cortex from AD brains. Oxysterol levels were quantified by isotope dilution mass spectrometry in ex vivo samples of AD brains classified by the Braak staging system. The figure gives the levels of: (A) two oxysterols of enzymatic origin, 24-hydroxycholesterol (24-OH) and 27-hydroxycholesterol (27-OH); (B) the oxysterols produced both enzymatically and non-enzymatically, 25-hydroxycholesterol (25-OH) and 7α-hydroxycholesterol (7α-OH); (C) the oxysterols of non-enzymatic origin, 7-ketocholesterol (7-K), 7β-hydroxycholesterol (7β-OH), 5α,6α-epoxycholesterol (α-epoxy) and 5β,6β-epoxycholesterol (β-epoxy), 4α-hydroxycholesterol (4α-OH) and 4β-hydroxycholesterol (4β-OH). Early AD (Braak stages I, II); late AD (Braak stages IV–VI). Control brain specimens: n=4; early AD specimens: n=5; late AD specimens: n=8. Brain tissues from the frontal and occipital cortex were analyzed separately. *P<0.05,**P<0.01, and ***P<0.001 vs controls; #P<0.05 and ##P<0.01 vs early AD.
Fig. 3
Fig. 3
Measurement of the expression levels of CYP46A1 and CYP27A1 in AD brain specimens. Gene expression was quantified by real-time RT-PCR in specimens at different stages of AD. Brain specimens of healthy subjects were taken as controls. Data, normalized to β2-microglobulin, are expressed as mean values±SD. Early AD (Braak stages I, II); late AD (Braak stages IV to VI). Control brain specimens: n=4; early AD specimens: n=5; late AD specimens: n=8. Brain tissues from the frontal and occipital cortex were analyzed separately. ***P<0.001vs controls; #P<0.05 and ###P<0.001 vs early AD.
Fig. 4
Fig. 4
Measurement of the expression levels of some inflammatory molecules in AD brain specimens. Gene expression of IL-1β, IL-6, IL-8, MCP-1 and MMP-9 was quantified by real-time RT-PCR in specimens at different stages of AD. Brain specimens of healthy subjects were taken as controls. Data, normalized to β2-microglobulin, are expressed as mean values±SD. Early AD (Braak stages I, II); late AD (Braak stages IV to VI). Control brain specimens: n=4; early AD specimens: n=5; late AD specimens: n=8. Brain tissues from the frontal and occipital cortex were analyzed separately. *P<0.05 and **P<0.01 vs controls; #P<0.05 and ##P<0.01 vs early AD.
Fig. 5
Fig. 5
Measurement of the expression levels of COX-2 and SIRT-1 in AD brain specimens. Gene expression of COX-2 and SIRT-1 was quantified by real-time RT-PCR in specimens at different stages of AD. Brain specimens of healthy subjects were taken as controls. Data, normalized to β2-microglobulin, are expressed as mean values±SD. Early AD (Braak stages I, II); late AD (Braak stages IV–VI). Control brain specimens: n=4; early AD specimens: n=5; late AD specimens: n=8. Brain tissues from the frontal and occipital cortex were analyzed separately. *P<0.05 and ***P<0.001 vs controls.
Fig. 6
Fig. 6
A hypothetical scheme for the involvement of oxysterols in the different stages of AD progression.

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