Upregulation of Glycolytic Enzymes, Mitochondrial Dysfunction and Increased Cytotoxicity in Glial Cells Treated with Alzheimer’s Disease Plasma

<div><p>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.</p></div

Summary of previous studies showing changes in acute phase proteins in Alzheimer’s disease.

<p>Summary of previous studies showing changes in acute phase proteins in Alzheimer’s disease.</p

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

<p><b>(A)</b> 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>(B)</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). <b>(C)</b> 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). <b>(D)</b> 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).</p

Cell viability of microglial cells after 48 hour incubation with human complement components (C1q, C1 inhibitor, C4, C5 and C9), both individually and in combination with each other; and a human complement standard containing complement components C1q, C2, C3, C4, C5, C6, C7, C8, C9 and factor B.

<p>Cell viability was determined by MTT assay of cell proliferation and intracellular NAD levels (for complement standard samples). Replicates included n = 6 for cell proliferation measurements and, n = 3 for NAD concentrations.</p><p>* p ≤ 0.05 vs Control,</p><p>** p ≤ 0.01 vs Control</p><p>Cell viability of microglial cells after 48 hour incubation with human complement components (C1q, C1 inhibitor, C4, C5 and C9), both individually and in combination with each other; and a human complement standard containing complement components C1q, C2, C3, C4, C5, C6, C7, C8, C9 and factor B.</p

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

<p>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.</p

Dysregulated proteins in glial cells treated with human control, MCI and AD plasma compared to FBS (non human serum control) following iTRAQ analysis.

<p>Cells were incubated with plasma in two 24 well plates, 3 wells for each of the plasma types were pooled from each plate to obtain two biological replicates for the 8-plex iTRAQ experiment. iTRAQ reporter ratios and p-values for altered proteins are shown for both replicates. Proteins found to be dysregulated in MCI and AD treated cells are shown in table. Proteins dysregulated only in cells treated with AD plasma are highlighted in bold. Full list of identified proteins can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116092#pone.0116092.s004" target="_blank">S2 Table</a>.</p><p>* p ≤ 0.05 vs Fetal Bovine Serum Control</p><p>Dysregulated proteins in glial cells treated with human control, MCI and AD plasma compared to FBS (non human serum control) following iTRAQ analysis.</p

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

<p>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.</p

Cell viability as measured by MTT absorbance (abs) at 570nm, LDH release and intracellular NAD levels of microglial cells after 48 hour incubation in pooled, non heat inactivated and heat inactivated MCI and AD plasma.

<p>* p ≤ 0.05 vs Control,</p><p>** p ≤ 0.01 vs Control</p><p>Cell viability was determined by measurement of cell proliferation, intracellular NAD levels and LDH activity in cell culture media and cell lysate homogenates. n = 9 (nine replicates) for cell proliferation measurements, n = 6 (six replicates) for NAD concentration and LDH activity. Plasma used for the measurements were obtained from the pooled plasma of 20 patients from each of the three groups (Control, MCI and AD) investigated. Three concentration levels of plasma were tested: 5%, 10% and 20% plasma as a percentage of total media volume.</p><p>Cell viability as measured by MTT absorbance (abs) at 570nm, LDH release and intracellular NAD levels of microglial cells after 48 hour incubation in pooled, non heat inactivated and heat inactivated MCI and AD plasma.</p