Fetal Alcohol SyndromeJuan-Carlos Andaluz

Normal 6 week infant FAS 6 week infant

Mouse EmbryosChild with FAS

Sanes 2012

EtOH-induced Oxidative

NMDA Receptor Alterations Membrane Damage & Lipid Peroxidation

Excitotoxic calcium mobilization

EdemaGlutamate Excitotoxicity Bax/ Cytochrome C/ Caspase

Neuronal Death

Schematic Model

NADPH oxidase (NOX)

Ermak et Al 2001

Schematic Model (continued...)

Starkov et Al 2004

Does Oxidative Stress induce both apoptosis as well as FAS-like

malformations ?

Oxidative Stress as the main pathway of pathogenesis in FAS

Dong et Al 2009

DPI treatment reduces OS-induced/NADPH Oxidase (NOX) malformations

C57BL/6J mice were mated for 2h. The time of vaginal plug detection was considered 0 days, 0h of gestation.

Pregnant mice in the experimental groups were administered 2 intraperitoneal doses of 2.9g/kg EtOH alone or 2 comparable doses of EtOH in combination with 1 4.0mg/kg dose of DPI (a NOX inhibitor).

The injections were given 4h apart, with the DPI & the first dose of EtOH administered on gestational day 9.

Control animals were injected with lactated ringer’s solution (which is isotonic with blood). The given treatment regimen produced a peak maternal blood alcohol level of ~ 500mg/dl 4.5h after the initial dose. Pregnant mice killed 6h after initial dose & whole embryos analyzedA) DPI B) 2.9g/kg EtOH C) Both EtOH & DPI D) Illustrates

excessive stain uptake in embryos exposed to EtOH alone.

% decrease

% decrease

% decrease

% decrease

Montoliu et Al 1995

Oxidative Stress (ROS) turns-on Apoptotic Cytochrome Pathway

The researchers used a non-fluorescent non-polar compound DCFH-DA (dichlorofluorescin-diacetate) that diffuses easily into cells to determine the effects of EtOH on the formation of ROS in Astrocytes. DCFH-DA is hydrolyzed by intracellular sterases (hydrolase enzymes) to a non-fluorescent polar derivative DCFH which stays trapped within cells. In the presence of intracellular Hydrogen Peroxide, DCFH is oxidized to the highly fluorescent compound DCF. Cellular fluorescence intensity was shown by the researchers to be directly proportional to the level of DCF formed inside cell.

Increased generation of ROS/products of lipid peroxidation AND decrease in GSH

Montoliu et Al 1995

MDA (marker of OS) & HNE are the main products that result from the decompostn of peroxides during lipid peroxidation.

The researchers measured the levels of GSH in cells exposed to 25/50mM ethanol & determined that EtOH exposure caused a dose-dependent decrease in GSH levels.

In this experiment, L-cysteine & L-cystine (both precursors to Glutathione) were added in addition to ethanol. When these amino acids were added to the astrocyte culture, they increased GSH synthesis and prevented the EtOH-induced

Conclusions

Evidence points to Oxidative Stress as the main biochemical mechanism of apoptosis & malformations observed in FAS.

EtOH-induced Oxidative Stress (via ROS) turns-on neuronal apoptotic machinery of Cytochromes and leads to decrease in defense machinery against ROS (GSH) (see diagram)

Inhibiting NOX (a producer of superoxide, a type of ROS) can prevent oxidative stress and malformations.

via C

alcium

via Calcium

Cohen-Kerem et Al 2002

Betzen et Al 2009

Interplay among Oxidative Stress (ROS) AND NMDA receptors

NR1 subunit (green) NR1 nuclei (red) stained with propidium iodine

At the protein level, NR1 expression was observed via immunochemistry

The researchers examined ROS-induced modifications in NMDA receptor function by using a fluorescent membrane-potential-sensitive dye “Bis Trimethine Oxonol”. They observed changes in ion influx mediated by NMDA receptor activation in bEnd3 Cells, which are mouse brain endothelial cells that express the essential NMDA receptor subunit NR1. The cells were treated with ROS for30 mins and after 72 hours they demonstrated that:

1) Exposure to ROS induces an up-regulation of functional NMDA Receptors & thus increases endothelial susceptibility to NMDA receptor stimuli. 2) Exposure to ROS leads to an increase in the expression of NR1 at the protein level, which results in enhanced glutamate excitotoxicity.