Treatment protocols.

<p>Cultures of aggregates were exposed to 1 mM GA and 3-OHGA at two time points representing different developmental stages of brain cell maturation (Protocols A and B). Metabolites were added 6 times every 12 hours (indicated by arrows) starting on DIV 5 in protocol A and on DIV 11 in protocol B (treatment days are indicated by black boxes) 12 hours after the change of the medium. Aggregates were harvested 5 hours after the last treatment at DIV 8 in protocol A and at DIV 14 in protocol B.</p

Effects of GA and 3-OHGA on neurons.

<p>(Left panel) Immunohistochemical staining for phosphorylated medium weight neurofilament (p-NFM) on cryosections of cultures derived from protocol A (DIV 8) and protocol B (DIV 14). Scale bar: 100 µm. (Right panel) Representative western blots with data quantification of whole-cell lysates for p-NFM for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of p-NFM are expressed as percentage of respective controls. The values represent the mean ± SD from 3 replicates taken from 2 independent experiments.</p

Evaluation of cell death after treatment with GA and 3-OHGA.

<p>(A; left panel) Immunohistochemical staining for cleaved caspase-3 (red signal). Scale bar: 100 µm. (A; right panel) Representative western blots with data quantification of whole-cell lysates for full length caspase-3 and the large fragment of cleaved (e.g. activated) caspase-3 for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of cleaved caspase-3 are expressed as percentage of respective controls. The values represent the mean ± SEM from 3 replicates taken from 2 independent experiments. (B) <i>In situ</i> cell death assay with TUNEL (green signal) and cleaved caspase-3 (red signal) on DIV 8 (protocol A). Merge of both signals leads to double-stained cells appearing in yellow. Scale bar: 100 µm. (C) LDH in culture medium of cultures from protocol A (DIV 8, above) and protocol B (DIV 14, below). Mean ± SD of 7 replicate cultures assessed by Student’s <i>t</i>-test; **p<0.01, *** p<0.001.</p

Additional file 1: Supplementary Methods. of Alzheimer disease pathology and the cerebrospinal fluid proteome

Table S1. Demographics and clinical characteristics of subjects removed from the statistical analyses. Table S2. Non-AD versus AD CSF biomarker profile group comparison after selection in all subjects of 26 proteins with LASSO. Table S3. Non-AD versus AD CSF biomarker profile group comparison after selection in subjects with cognitive impairment of 18 proteins with LASSO. Table S4. Correlation of CSF proteins with CSF Aβ1-42. Table S5. Correlation of CSF proteins with CSF tau. Table S6. Correlation of CSF proteins with CSF P-tau181. Table S7. Group comparisons of CSF protein measurements for AD versus non-AD CSF biomarker profiles in all subjects. Table S8. Group comparisons of CSF protein measurements for AD versus non-AD CSF biomarker profiles in subjects with cognitive impairment. Figure S1. Box-plots of CSF proteins (selected with LASSO analyses) for positive and negative CSF profiles of AD pathology in all subjects and subjects with cognitive impairment. Figure S2. Pairwise correlation heatmap of the 26 CSF proteins selected with LASSO for classification of non-AD versus AD CSF biomarker profiles for all subjects. Figure S3. Pairwise correlation heatmap of the 18 CSF proteins selected with LASSO for classification of non-AD versus AD CSF biomarker profiles for subjects with cognitive impairment. Figure S4. Correlations of CSF neurogranin and neuromodulin with CSF tau and P-tau181. Figure S5. Chord diagram of the relationships of 59 CSF proteins with CSF tau, P-tau181, and/or Aβ1-42. Figure S6. Venn diagrams of CSF proteins with significant group comparison differences between AD versus non-AD CSF biomarker profiles and those correlating with CSF Aβ1-42, tau, and P-tau181. Figure S7. Venn diagrams of CSF proteins selected with LASSO to classify non-AD versus AD CSF biomarker profiles and those correlating with CSF Aβ1-42, tau, and P-tau181. (DOCX 2575 kb