Blood methylomic signatures of presymptomatic dementia in elderly subjects with type 2 diabetes mellitus

Author version of article. The version of record is avilable from the publisher via doi: doi:10.1016/j.neurobiolaging.2014.12.023. Copyright © 2015 Elsevier Inc. All rights reserved.Available online 24 December 2014Due to an aging population, the incidence of dementia is steadily rising. The ability to identify early markers in blood, which appear before the onset of clinical symptoms is of considerable interest to allow early intervention, particularly in "high risk" groups such as those with type 2 diabetes. Here, we present a longitudinal study of genome-wide DNA methylation in whole blood from 18 elderly individuals with type 2 diabetes who developed presymptomatic dementia within an 18-month period following baseline assessment and 18 age-, sex-, and education-matched controls who maintained normal cognitive function. We identified a significant overlap in methylomic differences between groups at baseline and follow-up, with 8 CpG sites being consistently differentially methylated above our nominal significance threshold before symptoms at baseline and at 18 months follow up, after a diagnosis of presymptomatic dementia. Finally, we report a significant overlap between DNA methylation differences identified in converters, only after they develop symptoms of dementia, with differences at the same loci in blood samples from patients with clinically diagnosed Alzheimer's disease compared with unaffected control subjects.National Institute for Health Research (NIHR)Helen Bader FoundationLeroy Schecter FoundationBrightFocus Foundatio

Blood-Brain Barrier Cellular Responses Toward Organophosphates: Natural Compensatory Processes and Exogenous Interventions to Rescue Barrier Properties

Organophosphorus compounds (OPs) are highly toxic chemicals widely used as pesticides (e.g., paraoxon (PX)- the active metabolite of the insecticide parathion) and as chemical warfare nerve agents. Blood-brain barrier (BBB) leakage has been shown in rodents exposed to PX, which is an organophosphate oxon. In this study, we investigated the cellular mechanisms involved in BBB reaction after acute exposure to PX in an established in vitro BBB system made of stem-cell derived, human brain-like endothelial cells (BLECs) together with brain pericytes that closely mimic the in vivo BBB. Our results show that PX directly affects the BBB in vitro both at toxic and non-toxic concentrations by attenuating tight junctional (TJ) protein expression and that only above a certain threshold the paracellular barrier integrity is compromised. Below this threshold, BLECs exhibit a morphological coping mechanism in which they enlarge their cell area thus preventing the formation of meaningful intercellular gaps and maintaining barrier integrity. Importantly, we demonstrate that reversal of the apoptotic cell death induced by PX, by a pan-caspase-inhibitor ZVAD-FMK (ZVAD) can reduce PX-induced cell death and elevate cell area but do not prevent the induced BBB permeability, implying that TJ complex functionality is hindered. This is corroborated by formation of ROS at all toxic concentrations of PX and which are even higher with ZVAD. We suggest that while lower levels of ROS can induce compensating mechanisms, higher PX-induced oxidative stress levels interfere with barrier integrity

Albumin–Methotrexate Prodrug Analogues That Undergo Intracellular Reactivation Following Entrance into Cancerous Glioma Cells

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells

Schematic presentation of the proposed mechanisms involved in the chemical hydrolysis of methotrexate-amino derivatives covalently linked to carboxylate moieties of macromolecules (A) at acidic (B) and neutral (C) pH values.

<p>Schematic presentation of the proposed mechanisms involved in the chemical hydrolysis of methotrexate-amino derivatives covalently linked to carboxylate moieties of macromolecules (A) at acidic (B) and neutral (C) pH values.</p

Engineering a cysteine-specific MTX-containing reagent- Rates of hydrolysis following its conjugation to PEG₂₀-SH or to HSA at acidic pH.

<p>PEG₂₀-S-MAL-(CH₂)₂-CONH(CH₂)₆-NHCO-MTX and HSA-S-MAL-(CH₂)₂-CONH(CH₂)₆-NHCO-MTX were dialyzed against 1L of 1mM HCl and the decrease in the absorbance at 305nm in the dialysis tube was recorded applying the protocol as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158352#pone.0158352.g001" target="_blank">Fig 1</a>. Data are presented as mean±STDEV (n = 4).</p

Decrease in the absorbance of PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX with time during dialysis against 1mM HCl.

<p>PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX and PEG₄₀-CONH-(CH₂)₆-NHCO-MTX (0.2 μmole/ml of each) were dialyzed against one liter of 1mM HCl (pH 3.0). At the indicated time points, 30 μl aliquots were withdrawn from the dialysis tube, diluted 20 folds and their absorbance at 305 nm was monitored. Data are presented as mean±STDEV (n = 3).</p

MTX and Fmoc-L-glutamic acid structures.

<p>Illustrations of MTX (A) and Fmoc-L-glutamic acid (B).</p

An antiproliferative efficacy of BSA-MTX conjugates against MTX-sensitive CNS-1 cells.

<p>An antiproliferative efficacy of BSA-MTX conjugates against MTX-sensitive CNS-1 cells.</p