Cholesterol is essential for normal neuronal function, but high blood cholesterol is a risk factor for Alzheimer’s disease (AD). Astrocytes undertake the bulk of cholesterol synthesis in the brain; however, under stress conditions neurons can also synthesise cholesterol. Previous work showed that the neuronal DNA damage response is associated with lower cognitive function at the earliest stages of AD neuropathology, and this is associated with reduced expression of genes for cholesterol biosynthetic enzymes. The hypothesis for this study was that cholesterol dysregulation in neurons and astrocytes contributes to dementia and to the progression of AD. This study sought to determine the changes in expression of cholesterol biosynthetic enzymes in neurons and astrocytes with AD progression in post-mortem human tissue and in human neuronal and astrocytic monocultures.
Expression of HMG CoA reductase (HMGCR), the rate-limiting enzyme for cholesterol synthesis, and Sterol Regulatory Element Binding-Protein 2 (SREBP2), a key transcription factor regulating expression of cholesterol biosynthetic enzymes was examined by immunohistochemistry in the temporal cortex (Brodmann area 21/22) in an ageing population-based sample derived from a sub-cohort (n=99) of the Cognitive Function and Ageing Study (CFAS). Expression of HMGCR was predominantly associated with cortical pyramidal neurons within the cortex, although HMGCR expression was also associated with cells morphologically resembling astrocytes and oligodendrocytes in the cortex and white matter border regions of some cases. Quantification by determining HMGCR percentage immunoreactivity demonstrated population variation in HMGCR expression but no significant association with dementia status, AD neuropathology, oxidative DNA damage or neuroinflammation. SREBP2 was localised to neurons and astrocytes in temporal cortex and white matter regions.
Further work in post-mortem human tissue determined the expression of four key genes (HMGCR, CYP46A1, ABCA1 and SREBP2) involved with cholesterol biosynthesis and metabolism; in neuronal and astrocyte cell populations isolated from temporal cortex tissue by laser capture microdissection, from a sub-set of cases that included all Braak and Braak neurofibrillary tangle stages (n=32). Staging for tau pathology correlated with changes in neuronal gene expression of HMGCR, SREBP2 and ABCA1. Cholesterol concentration per mg of tissue was also determined in temporal cortex tissue using the Amplex Red cholesterol assay (n=63). Variation in tissue cholesterol concentration showed no significant association with dementia status, AD neuropathology, oxidative DNA damage or neuroinflammation. Furthermore, variation in HMGCR immunoreactivity and cholesterol biosynthetic gene expression in enriched samples of neurons and astrocytes isolated from temporal cortex did not correlate with temporal cortex cholesterol concentration. When tissue cholesterol concentration was categorically divided into tertiles and modelled using logistic regression analysis, moderate (OR:1.58, 95% CI: 0.39-6.43) and high (OR:1.44, 95% CI: 0.35-5.85) tissue cholesterol concentration was associated with dementia. However, the confidence intervals around these estimates were wide, indicating a high degree of uncertainty around the estimate.
In vitro work carried out in human neurons (LUHMES) and astrocytes (primary human foetal astrocytes) to determine the changes in expression of these genes in response to acute and a more chronic oxidative stress via a single and double exposure to H2O2. Both in vitro astrocytes and neurons expressed genes involved in cholesterol biosynthesis during neuronal proliferation, differentiation and in a post-mitotic state. Single and double exposure to 50 μM of H2O2 led to oxidative DNA damage in neurons and astrocytes, which persisted for 24 hrs and 96 hrs respectively. Preliminary data from stress experiments showed that the four key genes involved in cholesterol biosynthesis and metabolism (HMGCR, ABCA1, SREBP2 and APOE) were expressed in both cell types following a single and double exposure to H2O2. However; due to the COVID-19 pandemic, these experiments could not be completed.
In conclusion, this study has demonstrated that neurons possess the machinery to carry out de novo cholesterol biosynthesis in vivo and in vitro. Changes in gene expression of key regulators of cholesterol biosynthesis and metabolism alter with increasing tau pathology. However, these changes do not translate to changes in tissue cholesterol levels. This suggests that whilst there may be some dysregulation of cholesterol biosynthetic gene expression with increasing tau pathology, brain cholesterol itself is not altered by AD neuropathology and the association of altered brain cholesterol and dementia is weak.
