Upregulation of glycolytic enzymes, mitochondrial dysfunction and increased cytotoxicity in glial cells treated with Alzheimer's disease plasma

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder associated with increased oxidative stress and neuroinflammation. Markers of increased protein, lipid and nucleic acid oxidation and reduced activities of antioxidant enzymes have been reported in AD plasma. Amyloid plaques in the AD brain elicit a range of reactive inflammatory responses including complement activation and acute phase reactions, which may also be reflected in plasma. Previous studies have shown that human AD plasma may be cytotoxic to cultured cells. We investigated the effect of pooled plasma (n = 20 each) from healthy controls, individuals with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD) on cultured microglial cells. AD plasma and was found to significantly decrease cell viability and increase glycolytic flux in microglia compared to plasma from healthy controls. This effect was prevented by the heat inactivation of complement. Proteomic methods and isobaric tags (iTRAQ) found the expression level of complement and other acute phase proteins to be altered in MCI and AD plasma and an upregulation of key enzymes involved in the glycolysis pathway in cells exposed to AD plasma. Altered expression levels of acute phase reactants in AD plasma may alter the energy metabolism of glia.

Fig 1. Fractionation of non heat inactivated control plasma into protein and metabolite fractions and the effects of plasma treatment on cell proliferation.

Panel A: Fractionation of non heat inactivated control plasma into protein and metabolite fractions using PD10 column Panel B: Effect of these fractions on cell proliferation. Three replicates were performed. Plasma used for the measurements were obtained from the pooled plasma of 20 patients from each of the three groups (Control, MCI and AD). * p ≤ 0.01 vs Control, ** p ≤ 0.001 vs Control. Panel C: Images of microglia after 48 hour incubation with non heat inactivated 20% control plasma (left) and 20% AD plasma (right), showing increased toxicity and reduced cell proliferation in the AD plasma treated cells.

Fig 2. Effects of human plasma on cellular bioenergetics in a microglial cell line.

(A) Effect of human plasma on oxygen consumption rates (OCR) in a microglial cell line for 48 hours. *p<0.05 compared to non-treated cells (control); (n = 4 for each treatment group). (B) Effect of human plasma on extracellular acidification rates (ECAR) in a microglial cell line for 48 hours. *p<0.05 compared to non-treated cells (control); (n = 4 for each treatment group). (C) Effect of human plasma on the basal control ratio (BCR) in a microglial cell line for 48 hours. *p<0.05 compared to non-treated cells (control); (n = 4 for each treatment group). (D) Effect of human plasma on the uncoupling ratio (UCR) in a microglial cell line for 48 hours. *p<0.05 compared to non-treated cells (control); (n = 4 for each treatment group).

Fig 3. Chromatogram of fractionation using Hu6 column and 1D SDS/PAGE of these fractions.

Low abundant proteins are eluted first (first peak on chromatogram) and high abundant proteins are eluted after (second peak). Gel shows significant depletion of high abundant proteins in the low abundant fractions. Loading was 50 μg/lane. First and last lanes contained molecular weight markers. Each fraction was run in duplicate.

Fig 4. Glycolysis Pathway highlighting enzymes which were shown to be upregulated in cells treated with AD plasma.

Fig 5. The 27 proteins which were significantly deregulated in glia treated with AD plasma, but not deregulated in either control or MCI plasma treated glia (shown in Table 4) were analysed in STRING v9.1.

MCL clustering was used with the 2 clusters option picked and with the confidence view selected to display the strength of evidence for protein associations (panel A). Analysis of enrichment was also performed and the most significantly enriched biological process was glucose metabolic process (FDR p-value = 1.759x10E-8, with the 9 proteins involved in this process shown in panel B). Other distinct biological processes which were also significantly enriched included response to hydrogen peroxide (FDR p-value = 3.559x10E-2 with 4 proteins involved; ANXA1, PRDX1, PRDX2, CRYAB) and membrane to membrane docking (FDR p-value = 4.299x10E-2 with 2 proteins involved; MSN, EZR). Several molecular functions were also enriched, the most significant being RNA binding (FDR p-value = 5.340x10E-9 with 17 proteins involved shown in panel C). Another distinct and significantly enriched molecular function is thioredoxin peroxidase activity (FDR p-value = 9.220x10E-3 with 4 proteins involved; PRDX1, PRDX2). Several cellular components were also enriched, the most significant of these being extracellular vesicle exosome (FDR p-value = 5.019x10E-9 with 17 proteins involved as shown in panel D). Multiple other significantly enriched cellular components were also observed, and all enriched protein groups are shown in Table 5.