Metabolism-Based Gene Differences in Neurons Expressing Hyperphosphorylated AT8- Positive (AT8+) Tau in Alzheimer's Disease

Abstract

Metabolic adaptations in the brain are critical to the establishment and maintenance of normal cellular functions and to the pathological responses to disease processes. Here, we have focused on specific metabolic pathways that are involved in immune-mediated neuronal processes in brain using isolated neurons derived from human autopsy brain sections of normal individuals and individuals diagnosed as Alzheimer's disease (AD). Laser capture microscopy was used to select specific cell types in immune-stained thin brain sections followed by NanoString technology to identify and quantify differences in mRNA levels between age-matched control and AD neuronal samples. Comparisons were also made between neurons isolated from AD brain sections expressing pathogenic hyperphosphorylated AT8- positive (AT8+) tau and non-AT8+ AD neurons using double labeling techniques. The mRNA expression data showed unique patterns of metabolic pathway expression between the subtypes of captured neurons that involved membrane based solute transporters, redox factors, and arginine and methionine metabolic pathways. We also identified the expression levels of a novel metabolic gene, Radical-S-Adenosyl Domain1 (RSAD1) and its corresponding protein, Rsad1, that impact methionine usage and radical based reactions. Immunohistochemistry was used to identify specific protein expression levels and their cellular location in NeuN+ and AT8+ neurons. APOE4 vs APOE3 genotype-specific and sex-specific gene expression differences in these metabolic pathways were also observed when comparing neurons from individuals with AD to age-matched individuals.

Keywords: APOE and sex based changes; brain metabolism; differential gene expression; hyperphosphorylated tau; radical s-adenosyl domain 1.

Figure 1.

A: Differential mRNA expression between NeuN+ neurons captured using LCM from brain sections of individuals with AD compared to NeuN+ neurons captured from cognitively normal brain. Genes chosen for the study represented immune- modified metabolic pathways. Data are presented as a volcano plot where significantly increased mRNA expression levels at the p ≤ 0.05 level are shown in the upper right quadrant and genes with decreased mRNA levels at the p ≤ 0.05 level are shown in the upper left quadrant. MRNA level changes with a p-value of p < 0.1 are also indicated. Gene abbreviations are defined in Online Appendix Table 3. AD individuals: n = 20; Cognitively normal individuals: n = 11. B: Differential mRNA expression between LCM-captured AT8+ neurons from individuals with AD and NeuN+ neurons captured from cognitively normal brain. MRNA level changes with a p-value of p < 0.05 and p < 0.1 are noted. AD individuals: n = 20; Cognitively normal individuals: n = 11. C: Differential mRNA expression between AT8+ neurons from individuals with AD and NeuN+ neurons from the same AD brain. Cells were co-captured from an individual brain slice using double labeling techniques (n = 7). Genes with significantly (p ≤ 0.05) increased mRNA expression levels in AT8+ neurons compared to NeuN+ neurons in the same neuronal sample are shown in the upper right quadrant. Significantly decreased mRNA expression levels are shown for genes indicated in the left upper quadrant. MRNA level changes with a p-value of p < 0.05 and p < 0.1 are identified.

Figure 2.

Impact of APOE4 v APOE3 Genotype on Gene Expression Patterns. Volcano plot analysis shows differential mRNA expression changes in (A) NeuN+ neurons collected from cognitively normal individuals that expressed an APOE4/4 genotype (n = 4) compared to NeuN+ neurons from cognitively normal individuals that expressed an APOE3/3 genotype (n = 6); (B) AT8+ neurons collected from individuals diagnosed with AD and expressing either an APOE4/4 genotype (n = 10) compared to AT8+ neurons with an APOE3/3 genotype (n = 9); (C) APOE3/3 AT8+ neurons collected from individuals diagnosed with AD (n = 9) compared to APOE3/3 NeuN+ neurons from individuals diagnosed as cognitively normal (n = 4); and (D) AT8+ neurons expressing an APOE4/4 or APOE4/3 genotype from individuals diagnosed with AD (n = 10).

Figure 3.

Differential Gene Expression Based on Sex. Volcano plot analysis shows differential mRNA expression levels in NeuN+ and AT8+ neurons between females vs males. A: NeuN+ neurons from individuals diagnosed with AD; n = 9 females; 10 males; (B) AT8+ neurons from individuals diagnosed with AD; n = 3 female 3; 5 males; and (C) mRNA expression levels from NeuN+ cognitively normal females vs male (5 female; 3 male).

Figure 4.

Immunocytochemical Differences Between Autopsy Brain Sections Derived From Individuals With AD Compared to Cognitively Normal Individuals. Typical protein expression levels for genes associated with methylation processes in sections from frontal cortex autopsy samples of cognitively normal individuals (left panels) compared to similar samples from individuals diagnosed with AD (right panels). A and B: Representative immunohistochemical profile for human autopsy brain sections stained for Methylenetetrahydrofolate Reductase (MTHFR) from a cognitively normal individual (A) compared to an individual diagnosed with AD (B). Left panel- Male; 86 years; cognitively normal; APOE 3/3; CERAD 1; B&B–stage 1. Right panel – Female; 86 years; AD, APOE3/4; B&B-stage 4; AA mild infarcts. C and D: Representative immunohistochemical profile for human autopsy brain sections stained for Radical S-Adenosyl methionine domain containing 2 (RSAD2- also known as viperin) from a cognitively normal individual (left panel) compared to an individual diagnosed with AD (right panel). Left panel- Female, 84 years, cognitively normal; APOE3/3, CERAD 1; Right panel- Female, 74 years, AD, APOE4/4, B&B-stage 5. E and F: Representative immunohistochemical profile for human brain sections stained for Radical S-Adenosyl methionine domain containing 1 (RSAD1) from a cognitively normal individual (Left Panel) compared to an individual diagnosed with AD (Right Panel). Left panel- Male; 86 years; cognitively normal; APOE 3/3; CERAD 1; B&B–stage 1; Right panel- Male, 89 years; AD, APOE3/3, B&B-stage 5; AA- moderate.

Figure 5.

A to C: Co-localization of RSAD1 and NeuN immunoreactivities in a representative AD brain section. Fluorescent double labeling with RSAD1 and a neuron specific antibody (NeuN) was used to observe the overlap between RSAD1 and the population of neurons from a section of AD brain. A: Localization of neurons showing RSAD1 immunoreactivity (green). B: Localization of all neuronal sub types using immunoreactivity for the NeuN antibody (red). C: The presence of RSAD1 immunoreactivity co-localized with NeuN immnostaining on a sub-population of neurons (for example, (C) lower left). However, all NeuN+ neurons are not double labeled with RSAD1 immunostain (for example, (C) upper right). Pathology-Female; hippocampus, AD Brock Stage 5, 88 years. D: Double labeling was used to identify RSAD1+ cells (Black/gray stain) from 4G8+ amyloid containing neuritic plaques (Red/orange stain). E and F: Western blot was used to quantitate changes in RSAD1 protein levels (E) compared to a protein control (Vinculin, (F)). Samples were obtained from isolated brain sections from cognitively normal individuals compared to individuals with AD. ** p<0.01, n = 5 control (non-AD) and 11 AD brain samples.

Figure 6.

Representative Metabolic Pathway Interactions for Proteins Involved With Arginine, Ornithine, Methionine and Redox-Based Metabolic Pathways. Dashed box—Proteins affected in AT8+ neurons compared to non-AT8+/NeuN+ neurons. Dashed Line—RSAD1 activity in this pathway is based on its currently known cell-based effects and is estimated here.