Feb. 10, 2003 DURHAM, N.C. -- A Duke University Medical Center neurobiologist has identified key mechanisms by which the intricate "protein machines" that govern the strength of connections among neurons build and remodel themselves to adjust those connections.
Such remodeling of the connections, called synapses, is central to the establishment of brain pathways during learning and memory, said the scientists. Also, malfunction of the synaptic machinery might well play a fundamental role in the pathology of neurodegenerative disorders including Parkinson's and Alzheimer's diseases.
The findings were reported in the advanced online version of the March 2003 Nature Neuroscience by neurobiologist Michael Ehlers.
Said Bill Thies, Ph.D., vice president, medical and scientific affairs of the Alzheimer's Association, a sponsor of the research "The discovery of the earliest events in Alzheimer's disease is very important to understanding the disease. This paper on activity-dependent synaptic organization and disorganization opens an interesting path to the earliest perturbations of Alzheimer's."
The work was also supported by the National Institutes of Health and other private foundations.
In the Nature Neuroscience paper, Ehlers reported extensive experiments revealing the function of a structure known as the "post-synaptic density" (PSD). The PSD is so named because it is a thickening of the membrane at the connection point between neurons, where one neuron receives biochemical signals called neurotransmitters from its neighbor. Such neurotransmitters are the means by which one neuron triggers the receiving neuron to launch a nerve impulse.
"The post-synaptic density has been known for decades as a distinctive structure readily visible under an electron microscope," said Ehlers, who is an assistant professor of neurobiology. "Also, many of its protein components have been identified -- including neurotransmitter receptors, scaffolding proteins, signaling enzymes and adhesion molecules. So, it was clear that this was an important specialized machine for receiving the chemical signal from the pre-synaptic nerve cell."
Also, said Ehlers, experiments by other researchers had shown that the PSD significantly alters its shape in response to the kind of neural activity that takes place during learning. They had also established that the neurotransmitter receptors in the PSD move in and out of the membrane during such remodeling. "But what past work has not shown is how the many components of this machine behave together," said Ehlers. "Our goal was to take a step back and look at patterns of protein accumulation and loss, rather than examining one molecule at a time -- to provide a molecular fingerprint if you will." The key to achieving such broad insights into PSD remodeling, said Ehlers, was to explore the gain or loss of a multitude of known PSD proteins, and not just one or two. Thus, in his experiments, Ehlers developed a "protein expression profiling" technique to isolate and measure the levels of some 30 proteins in the PSDs of cultured rat embryo neurons. This mass analysis revealed a distinct pattern of protein turnover, he said.
"We found that a significant percentage of the major PSD protein components moved up and down with neural activity," he said. "And surprisingly, they didn't behave independently, but moved as groups or ensembles, with a whole set going up or down in response to activity. Even more notable was that we saw the exact mirror image pattern in behavior when neural activity was blocked." According to Ehlers, such a discovery could have a profound impact on scientists' ability to understand the structure and function of the intricately complex protein machine that is the PSD.
"These findings allow us to begin making testable predictions about the functional networks of proteins in these synaptic complexes," he said. "For example, we believe that there are probably some master organizers in the PSD that recruit or organize large subsets of these proteins. Now, we can search for those master molecules." Further, "by providing a molecular fingerprint of the functional state of the synapse, we can now begin to compare patterns as the brain develops, ages, and learns, as well as in disease states such as Alzheimer's disease, or even across individuals with different experiences and environmental exposures."
Another surprise, Ehlers found, was the mechanism behind the turnover of such proteins. In his experiments, Ehlers explored whether the cell's protein "garbage collection" system might be involved in the turnover. In this system, proteins targeted for destruction are tagged with a molecule called ubiquitin and transported to a shredding complex called the proteasome for destruction. Ehlers' studies revealed that this system was required for normal turnover of PSD proteins. Ehlers also showed that the ubiquitin-proteasome system affected specific metabolic pathways in the neurons that are known to be involved in the changes in synaptic connections associated with learning and memory. "The prevailing model for long-term plastic change at synapses has been that genes are switched on to make new proteins and incorporate them into the synapse," said Ehlers. "Much, much less appreciated has been the fact that proteins must also be removed from the structure. And what we found was that this highly regulated removal is a key part of the remodeling of the PSD."
Also startling, said Ehlers, was the high base level of remodeling of the PSD. "I found that neurons in these cultures replace the content of this signaling machine multiple times a day," said Ehlers. "And if this recapitulates what's going in the mammalian brain, this means that synapses are completely turning over all of their constituents multiple times a day – a stunning finding."
Neuroscientists have long been intrigued in how the brain changes with learning and experience, a phenomenon called plasticity. Yet, as Ehlers points out, "perhaps we need to think more closely about how connections in the brain remain stable in the face of such incredible ongoing turnover."
"In fact, when I was doing these experiments, I anticipated a turnover on the order of days, so I took my first time measurement at about a day and found no protein label left in the sample. I thought the experiment had failed until I decided to do measurements at earlier time points."
According to Ehlers, the new insights into the PSD his studies allow could have important implications for understanding neurodegenerative diseases including Parkinson's and Alzheimer's diseases.
"Many of these diseases have as their hallmark pathology abnormal deposits in the brain, many of which show high levels of ubiquitin," he said. "For example, there are rare familial versions of Parkinson's that arises from mutations in genes that regulate the attachment of ubiquitin to proteins. So, these findings might give new insights into how such mutations affect the brain. Such findings might also shed light in the subtle pathological changes in synaptic connections that eventually give rise to Alzheimer's disease," he said.
"There is an increasing appreciation of the idea that before the neurological damage from Alzheimer's disease becomes apparent, there may be subtle synaptic defects that cause only a mild cognitive decline," said Ehlers. "This study showing the central relationship between the ubiquitin system and synaptic organization gives us a research pathway to trace the possible origin of these subtle defects. And it is my hope that such basic insights will lead to therapies to remedy the defects early."
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