Molecular Profiling Reveals Diversity of Stress Signal Transduction Cascades in Highly Penetrant Alzheimer's Disease Human Skin Fibroblasts

The serious and growing impact of the neurodegenerative disorder Alzheimer's disease (AD) as an individual and societal burden raises a number of key questions: Can a blanket test for Alzheimer's disease be devised forecasting long-term risk for acquiring this disorder? Can a unified therapy be devised to forestall the development of AD as well as improve the lot of present sufferers? Inflammatory and oxidative stresses are associated with enhanced risk for AD. Can an AD molecular signature be identified in signaling pathways for communication within and among cells during inflammatory and oxidative stress, suggesting possible biomarkers and therapeutic avenues? We postulated a unique molecular signature of dysfunctional activity profiles in AD-relevant signaling pathways in peripheral tissues, based on a gain of function in G-protein-coupled bradykinin B2 receptor (BKB2R) inflammatory stress signaling in skin fibroblasts from AD patients that results in tau protein Ser hyperphosphorylation. Such a signaling profile, routed through both phosphorylation and proteolytic cascades activated by inflammatory and oxidative stresses in highly penetrant familial monogenic forms of AD, could be informative for pathogenesis of the complex multigenic sporadic form of AD. Comparing stimulus-specific cascades of signal transduction revealed a striking diversity of molecular signaling profiles in AD human skin fibroblasts that express endogenous levels of mutant presenilins PS-1 or PS-2 or the Trisomy 21 proteome. AD fibroblasts bearing the PS-1 M146L mutation associated with highly aggressive AD displayed persistent BKB2R signaling plus decreased ERK activation by BK, correctible by gamma-secretase inhibitor Compound E. Lack of these effects in the homologous PS-2 mutant cells indicates specificity of presenilin gamma-secretase catalytic components in BK signaling biology directed toward MAPK activation. Oxidative stress revealed a JNK-dependent survival pathway in normal fibroblasts lost in PS-1 M146L fibroblasts. Complex molecular profiles of signaling dysfunction in the most putatively straightforward human cellular models of AD suggest that risk ascertainment and therapeutic interventions in AD as a whole will likely demand complex solutions

Mediators and modulators of fibroblast signaling in Alzheimer's Disease.

<p>A. Schematic of the MAPK pathway. The MAPK pathway functions by sequential phosphorylation events: MAP3K→MAP2K→MAPK→substrates. The three MAPK modules consisting of JNK1/2, p38 and ERK1/2 are both indicators and mediators in stress responses. MAPKs regulate nuclear target genes through phosphorylation of multiple transcription factor substrates (TF) as well as membrane and cytoskeletal protein targets. B. Schematic of PS-1 and sites of familial AD mutations. Presenilin-1 (PS-1) functions as a key component in the gamma-secretase complex along with participants PS-2, nicastrin (NCT), anterior pharynx-defective phenotype 1 (APH-1) and PS enhancer-2 (PEN-2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004655#pone.0004655-Kim1" target="_blank">[38]</a>. PS-1 has multiple transmembrane (TM) domains and the 3 sites of Familial AD (FAD) mutations investigated are positioned within their respective domains.</p

Immunoreagents.

<p>WB = Western blotting, IF = Immunofluorescence.</p

JNK promotes a survival function in normal fibroblasts that is lost in PS-1 (M146L) AD fibroblasts.

<p>PS-1 (M146L) AD and control fibroblasts were pre-treated with DMSO vehicle control or SP 600125 inhibitor in buffer, oxidatively stressed with 250 µM H₂O₂ for 60 min and re-fed with media for 0–10 hr, then methanol-fixed and Hoechst stained as previously described. (A) Quantitation of apoptosis due to H₂O₂. The No Inhibitor condition (NI) represents buffer without DMSO or SP 600125, with or without H₂O₂ during the 60 min incubation. The number of apoptotic nuclei was quantitated as a percentage of condensed nuclei to total nuclei per field. Graph shows mean±S.E. Statistical analysis was performed via t-test with *p-value<0.05; **p<0.005*** and p<0.0005; n = 3. Black * and *** brackets compare the percentage of apoptotic cells between AD and control fibroblast lines in the presence of DMSO or SP600125. Purple * and ** brackets compare the percentage of apoptotic cells within the control fibroblast line between the DMSO and SP600125 treatments. (B) Confirmation of SP600125 inhibition of JNK. PS-1 (M146L) AD fibroblasts pre-treated with DMSO or SP600125 for 45 min and then with 250 µM H₂O₂ for 0–20 min were immunoblotted and analyzed for phospho-JNK and total JNK by ECL and Odyssey respectively, normalized to GAPDH as a loading control.</p

Hydrogen peroxide treatment results in an increased proportion of apoptotic nuclei in PS-1 (M146L) AD fibroblasts.

<p>PS-1 (M146L) AD and control fibroblasts treated with 500 µM H₂O₂ and re-fed with media for 0–25 hr were methanol-fixed, Hoechst stained, and the number of total and apoptotic nuclei quantitated. Graph shows percentage of condensed nuclei relative to total number of nuclei, mean±S.E. Statistical analysis was performed via t-test with ***p<0.0005; n = 3.</p

Gamma-secretase inhibitor Compound E affects BK-dependent p38 activation in PS-1 (M146L) AD fibroblasts but not ERK activation in Trisomy 21 fibroblasts.

<p>Trisomy 21 and PS-1 (M146L) AD fibroblasts plus normal control human skin fibroblasts were treated with Gamma secretase inhibitor Compound E (10 nM) or DMSO vehicle control for 24 hr and then stimulated with 250 nM BK for 0–30 min. Cell lysates were immunoblotted and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004655#pone-0004655-g006" target="_blank">Figures 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004655#pone-0004655-g007" target="_blank">7</a>, by Odyssey for ERK (A,B) and by chemiluminesence for p38 (C,D). Activity levels were normalized to tubulin as a loading control. ERK activation was quantified as a percentage of basal activation in buffer-treated cells (0 min) pre-treated with DMSO or Compound E (panel B). In panel D p38 activation was quantified as O.D. values as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004655#s4" target="_blank">Materials and Methods</a>. ERK activity phenotypes are graphically represented in control fibroblasts compared with AD pre-disposed Trisomy 21 fibroblasts (B), and p38 activity phenotypes are graphically represented in control and PS-1 (M146L) AD fibroblasts (D). All graphs show mean±S.E. Statistical analysis was performed via t-test with **p-value<0.005; n = 3.</p

Hydrogen peroxide treatment results in heightened caspase-3 activation in PS-1 (M146L) AD fibroblasts.

<p>PS-1 (M146L) AD and control fibroblasts were treated with 500 µM H₂O₂ for 60 min and then re-fed with media for 0–20 hr to permit a post stress time window. Cells were methanol-fixed and analyzed by immunocytochemistry for cleaved caspase-3 with Alexa-488 labeled secondary antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004655#s4" target="_blank">Methods</a>. Representative images of fields uniformly containing 35–40 cells were captured at time points designated (A–F). PS-1 (M146L) AD fibroblasts; (G–I) control fibroblasts. Representative phase contrast images of (J) control and (K) PS-1 (M146L) AD cells show comparable growth properties and cell densities at time of testing for oxidative stress responses. (L) Alexa 488 fluorescence intensity of cleaved caspase-3 immunostaining in PS-1 (M146L) fields A–F above and control fields G–I was quantitated via the MacBiophotonics Image J analysis program (<a href="http://www.macbiophotonics.ca/downloads.htm" target="_blank">www.macbiophotonics.ca/downloads.htm</a>). Bar graph represents 2 independent experiments' mean and S.E.</p

Gamma-secretase inhibitor Compound E restores normal BK-dependent ERK activation in PS-1 (M146L) AD fibroblasts.

<p>PS-1 (M146L) AD and normal control human skin fibroblasts were treated with Gamma secretase inhibitor Compound E (1 or 10 nM) or DMSO vehicle control for 24 hr and then stimulated with 250 nM BK for 0–30 min. Cell lysates were immunoblotted for ERK as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004655#pone-0004655-g006" target="_blank">Figure 6</a> and analyzed by Odyssey for ERK activation normalized to Tubulin as loading control. A representative blot is shown in (A). ERK activation was quantified as a percentage of basal activation in buffer-treated cells (0 min) pre-treated with DMSO or Compound E. ERK activity phenotypes are graphically represented in control fibroblasts compared with PS-1 (M146L) AD fibroblasts (B). All graphs show mean±S.E. Statistical analysis was performed via t-test with *p-value<0.05; n = 3.</p

PS-1 (M146L) AD fibroblast line displays persistent BK-B2R modulation upon PKC activation.

<p>PS-1, PS-2 and control non-AD fibroblasts (CTRL) were treated with 25 nM PMA for the indicated times. B2 receptors in non-ionic detergent solubilizates were detected by immunoblotting and chemiluminescence detection. Results are representative of 3 experiments for PS-1 and control and 4 for PS-2.</p

JNK inhibitor SP 600125 enhances oxidative stress apoptosis in control fibroblasts.

<p>PS-1 (M146L) AD and control fibroblasts were pre-treated with DMSO vehicle control or JNK inhibitor SP 600125 (25 µM) for 45 minutes in buffer. Oxidative stress was induced with 250 µM H₂O₂ for 60 min and cell layers were then re-fed with media plus vehicle control or JNK inhibitor for 0–10 hr. Cells were methanol-fixed and Hoechst stained to label nuclei and distinguish between normally sized nuclei versus condensed apoptotic nuclei. The No Inhibitor column (Panels A, D, G, and J) represents cells incubated in buffer without DMSO or SP 600125 but in the presence or absence of H₂O₂ during the initial 60 min incubation. Hoechst staining is displayed in grey for PS-1 (M146L) AD fibroblasts (A–F) and control fibroblasts (G–L). Representative captured images shown display signs of apoptosis with H₂O₂ treatment at designated time points.</p