Neuronal DNA damage response-associated dysregulation of signalling pathways and cholesterol metabolism at the earliest stages of Alzheimer-type pathology

Abstract

Aims: Oxidative damage and an associated DNA damage response (DDR) are evident in mild cognitive impairment and early Alzheimer's disease, suggesting that neuronal dysfunction resulting from oxidative DNA damage may account for some of the cognitive impairment not fully explained by Alzheimer-type pathology.

Methods: Frontal cortex (Braak stage 0-II) was obtained from the Medical Research Council's Cognitive Function and Ageing Study cohort. Neurones were isolated from eight cases (four high and four low DDR) by laser capture microdissection and changes in the transcriptome identified by microarray analysis.

Results: Two thousand three hundred seventy-eight genes were significantly differentially expressed (1690 up-regulated, 688 down-regulated, P < 0.001) in cases with a high neuronal DDR. Functional grouping identified dysregulation of cholesterol biosynthesis, insulin and Wnt signalling, and up-regulation of glycogen synthase kinase 3β. Candidate genes were validated by quantitative real-time polymerase chain reaction. Cerebrospinal fluid levels of 24(S)-hydroxycholesterol associated with neuronal DDR across all Braak stages (rs = 0.30, P = 0.03).

Conclusions: A persistent neuronal DDR may result in increased cholesterol biosynthesis, impaired insulin and Wnt signalling, and increased GSK3β, thereby contributing to neuronal dysfunction independent of Alzheimer-type pathology in the ageing brain.

Keywords: Ageing brain; Alzheimer's; DNA damage response; dementia; microarray; neurones.

Figure 1

Laser capture microdissection (LCM) of neurones. Toludine blue positive neurones were isolated (a) before and (b) after LCM, as indicated by the arrows.

Figure 2

Gene expression analysis of cases with a high neuronal DNA damage response (DDR). (a) Principal component analysis of microarray data. One sample outlier in the low‐DDR group (indicated by the arrow) and one Pick's disease case in the high DDR group (indicated by the arrow head) were removed from subsequent analysis. (b) Heat map depicting up‐regulated (green) and down‐regulated (red) gene expression changes (P < 0.001). A high neuronal DDR was associated with the up‐regulation of 1690 genes and the down‐regulation of 688 genes.

Figure 3

Immunohistochemical assessment of protein expression encoded by candidate genes. Neuronal expression of (a) neurofilament light (NFL), (b) IRS1, (c) glycogen synthase kinase 3β (GSK3β), (d) Wnt‐3a and (e) HMG–CoA reductase (HMGCR). (f) sterol regulatory element binding proteins (SREBP)2 was associated with both neurones (arrow) and astrocytes (arrow head). Scale bar represents 50 μm.

Figure 4

Protein expression encoded by candidate genes. Western blot analysis of total protein extracts demonstrated significant up‐regulation of neurofilament light (NFL) (68 kDa) and down‐regulation of Wnt‐3a (39 kDa) in cases with a high neuronal DNA damage response (DDR). **P < 0.01. AU, arbitrary units.

Figure 5

Cholesterol turnover correlates with increased levels of both astrocyte and neuronal DNA damage response (DDR) and inversely associates with Braak stage. Cerebrospinal fluid (CSF) levels of 24(S)‐OHC (a) inversely associated with increasing Braak and Braak stage, and positively associated with both (b) an astrocytic DDR (r _s = 0.43, P = 0.001)and (c) a neuronal DDR (r _s = 0.30, P = 0.033). Braak group 1 = Braak and Braak stage 0–II, Braak group 2 = Braak and Braak stage III–IV, Braak group 3 = Braak and Braak stage V–VI. AU = arbitrary units.