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[AB.sub.42] has potential direct and indirect effects on mitochondria leading to Alzheimer's disease.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases impacting the elderly; therefore, it is imperative to find out what factors may be the cause. From pathologic reviews in human brain, hallmarks of AD include extracellular plaques and intracellular neurofibrillary tangles (breakdown of cytoskeleton) in parts of the brain involved with cognition and memory. The extracellular plaques are formed in part from aggregates of the amyloid beta protein (AB) secreted by nerve ceils, which may play a causative role in AD. Plaques and tangles seen in AD may be secondary to the actual intracellular events induced by AB fragments within ceils which lead to cell death and AD.

AB originates from the amyloid precursor protein (APP), which is synthesized in the rough endoplasmic reticulum (RER). Enzymes, specifically the beta and gamma secretases, cleave APP to a number of short fragments, primarily 40 or 42 amino acids in length. The 42 amino acid long fragment (AB42) is believed to be the neurotoxic form. Research has shown that interaction of [AB.sub.42] with intracellular membranes affords the peptide opportunity to induce apoptotic cell death, particularly in hippocampus and cerebral cortex. Such pathology leads to changes commonly manifested in AD including memory loss, personality changes, and cognitive deterioration.

Using an AD rat model, our experiments have demonstrated the presence of [AB.sub.42] in the endoplasmic reticulum, on mitochondria and the nucleus. The focus was to determine how [AB.sub.42] interacts with these various intracellular membranes. Of particular interest are mitochondria, which regulate metabolic respiration via ATP production, and control apoptosis by either facilitating or inhibiting this form of cell death.

Chloroquine challenge was used to raise the intracellular levels of [AB.sub.42] in rat brain, and control brains were used to compare results. Differential centrifugation of brain homogenates on a percoll gradient was used to isolate mitochondria. Mitochondria were stained with Mitotracker red (chloromethyl-X-rosamine, CMX), then viewed by confocal microscopy to detect CMX fluorescence. High CMX fluorescence indicates the viability of mitochondria (an indirect measure of normal transmembrane potential). Reduction in CMX response under experimental conditions (increased intracellular [AB.sub.42]) suggests that [AB.sub.42] damages the mitochondria (loss of transmembrane potential) a condition that favors apoptosis.

To test for the induction of apoptosis by intracellular [AB.sub.42]' similarly isolated mitochondrial preparations were fixed in 4% paraformaldehyde/0.5% glutaraldehyde and embedded in Lowicryl. Thin sections (60nm) cut from the experimental and control mitochondrial pellets were immunolabeled with antibodies that target antigens active in apoptotic cascades. The antigens Bax, cytochrome c and caspase 3 were labeled with primary antibodies and secondary antibodies conjugated to gold particles (18nm) and viewed with the electron microscope (EM immunogold technique). Gold particles observed in electron micrographs of experimental mitochondria suggest that [AB.sub.42] acts on mitochondria to induce activation of these apoptotic favoring compounds. [AB.sub.42] induces lipid peroxidation and one product of this process, 4-Hydroxynonenal (4-HNE), directly attacks mitochondria, and thereby plays a central role in oxidative stress and cell death.

The outcome of this research thus far has shown that the [AB.sub.42] fragment is found inside of hippocampal pyramidal ceils, specifically on mitochondria, and induces apoptosis through various means. The research is discovering possible mechanisms of how [AB.sub.42] kills these cells, whose loss in humans contributes to the profound memory impairments of AD.

Katherine M. Splichal, Shane M. Meyer, Krysta Mann, and Garl K. Rieke

Department of Anatomy and Cell Biology, University of North Dakota, Grand Forks, ND 58202
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Title Annotation:A. Rodger Denison Student Research Competition: COMMUNICATIONS: UNDERGRADUATE DIVISION
Author:Splichal, Katherine M.; Meyer, Shane M.; Mann, Krysta; Rieke, Garl K.
Publication:Proceedings of the North Dakota Academy of Science
Geographic Code:1USA
Date:Apr 1, 2004
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