Alzheimer's disease begins with mild dementia, and is ultimately terminal. It afflicts millions around the world, and there is currently no cure.
It is known that Alzheimer's disease is associated with the development of protein plaques that inhibit the function of neurons in the brain. Understanding the molecular details of this inhibition may help lead to a cure.
Nelson Arispe (Uniformed Services University of the Health Sciences, Maryland) and coworkers have shed light on the molecular basis for Alzheimer's disease. They have added weight to the hypothesis that the toxicity of protein plaques is due to their capacity to punch holes in neuron cell membranes.
The hypothesis.
The scientists knew that amyloid beta protein plaques form channels in neuron cell membranes. They further knew that two chemical functionalities known as histidine are positioned at the entrance of and line the path through the channels.
They reasoned that molecules which form a chemical bond with these two histidine functionalities at the entrance of amyloid beta protein channels would disrupt the channels. This would prevent amyloid beta toxicity, and could possibly serve as the basis for a medical treatment for Alzheimer's disease.
Testing the hypothesis in artificial constructs.
This hypothesis was first tested in an artificial cell membrane known as a planar lipid bilayer. These constructs are widely used as primitive, yet functional and addressable, mimics of cell membranes. Amyloid beta protein channels were incorporated into these particular constructs.
A four times greater amount of cesium ions was present on one side of the artificial cell membrane than the other side. Therefore, by diffusion from a region of higher ion concentration to one of a lower ion concentration, if the amyloid beta protein channels are open, there will be a cesium ion flux through the artificial cell membrane.
Conversely, if the amyloid beta protein channels are closed, there will not be a cesium ion flux. The flux, or lack of flux, of cesium through the channels can be measured electrically.
Therefore, if a molecule blocks the channels, by chemically binding to the histidine functionalities, there will not be a flux of cesium ions through the artificial cell membranes. This is a convenient method to report on amyloid beta protein channel disruption.
The scientists found that nickel ions and imidazole molecules, both of which are known to bind to histidine functionalities, irreversibly block amyloid beta protein channels. In the case of imidazole, the five most commonly observed electrical currents disappeared after 106 seconds.
Testing the blocking mechanism.
At ths point, the scientists knew that imidazole molecules block amyloid beta protein channels. Since it is well-known that imidazole forms a strong chemical bond with histidine, it is reasonable to suspect that imidazole blocks amyloid beta protein channels by forming a chemical bond with the histidine functionalities in the channel.
This hypothesis can be tested by chemically altering imidazole molecules, by either enhancing or hindering their chemical affinity for histidine, and observing the channel blocking properties of the molecules. This is what the scientists did next.
The most effective blocking molecule that the scientists evaluated was one that possessed a large number of imidazole functionalities.
Possessing four imidazole functionalities enabled a molecule to block the channels by 50% in 15 seconds. In contrast, a molecule that possessed only two imidazole functionalities required 125 seconds (8 times longer) to achieve the same level of channel blocking.
Additionally, chemically modifying a successful channel blocking molecule, by incorporating a methyl functionality into the imidazole subunit, eliminated channel blocking ability. These experiments demonstrate the specific efficacy of imidazole in blocking amyloid beta protein channels.
Testing the hypothesis in cells.
So far, the scientists have tested amyloid beta protein channel blocking in artificial constructs. They also performed channel blocking experiments in cells, experiments which are more medically relevant.
The scientists tested the ability of channel-blocking molecules to prevent death in cells possessing amyloid beta protein channels. Two methods were used to quantify cell viability.
One of the methods for quantifying cell viability was based on XTT, the simple abbreviation for a molecule of the much longer formal name 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide. This molecule is processed by metabolically active (living) cells into a colored product. When more colored product is detected, more of the cells are alive.
The second method for quantifying cell viability was based on measuring the release of the enzyme lactate dehydrogenase from the cells. More release of the enzyme from the cells means that the cells are leaking, and are not alive.
Three cell types, all incorporating amyloid beta protein channels, were tested for protection from death by the channel-blocking molecules studied previously in the artificial cell membranes. In all cases, molecules that blocked channels in artificial cell membranes kept cells alive, and molecules that did not block channels in artificial cell membranes did not keep cells alive.
Curing Alzheimer's disease.
Nelson Arispe and coworkers (and other scientists) have previously found evidence that amyloid beta proteins form channels in cell membranes. This research further adds to the evidence that disrupting these channels, using imidazole molecules, helps to prevent cell death.
These experiments were performed in artificial constructs and in model cells. Therefore, it cannot necessarily be said that a cure for Alzheimer's disease is in the making. However, these results do show promise, and should be welcomed and further investigated by doctors, who currently are unable to either cure or prevent this fatal disease.
for more information:
Arispe, N.; Diaz, J. C.; Flora, M.
Efficiency of histidine-associating compounds for blocking
the Alzheimer's Aβ channel activity and cytotoxicity.
Biophys. J. 2008, 95, 4879-4889.