The Latest Results for a New Theory

By Jason Socrates Bardi

Last fall at one of several poster sessions at the 2004 Society for Neurosciences meeting in San Diego, thousands of posters on display reported hypotheses, data, conclusions, and images of all matters related to the brain—everything from the results of a high school neuroscience art contest in Nova Scotia to a study in which researchers mapped which parts of the brain are active when a person finds out he/she has been given a raise at work.

One of these posters was presented by several members of the Department of Neurobiology at The Scripps Research Institute and titled, "Altered mRNA granule composition and translation factor regulation in FMR1 knockout mice." Its authors, Research Associate Armaz Aschrafi, Professor Bruce Cunningham, and Assistant Professor Peter Vanderklish, were on hand to answer questions posed by conference attendees, mostly neuroscientists and at least one writer.

Recently, this same writer had occasion to return to the subject when an article by the same group appeared on the web site of the journal Proceedings of the National Academy of Sciences. The article, like the poster, was about a new theory, which Dr. Mark Bear of the Massachusetts Institute of Technology termed “the mGluR theory.”  The mGluR theory concerns fragile X syndrome—the most commonly inherited form of mental retardation in the United States.

A Disease Caused by a Single Inherited Gene

Fragile X syndrome is an incurable genetic condition that affects about one in 4,000 males and one in 8,000 females of all races born in the United States, according to statistics compiled by the U.S. Centers for Disease Control and Prevention. In addition to being more common among boys than girls, the condition affects boys more severely.

Children born with fragile X syndrome often develop learning disabilities, behavioral problems, autistic behavior, emotional problems, characteristic physical traits, and mild to severe mental retardation. Boys born with fragile X syndrome often have severe mental retardation. According to the National Institute of Child Health and Human Development, more than 80 percent of males with fragile X have an I.Q. of 75 or less.

The disease was first described in 1969, and like many causes of mental retardation, it affects neurons in the brain—including those in the hippocampus and cortex that are important for memory and information processing. In the early 1990s, fragile X syndrome was mapped to a single gene on the X chromosome called Fmr1. The protein that the Fmr1 gene encodes was dubbed FMRP, an acronym for fragile X mental retardation protein.

The mutations linked to fragile X syndrome are what are known as “triplet repeats” of DNA that appear in the non-coding region of Fmr1—the DNA immediately before the part of the Fmr1 gene that is expressed as FMRP. There, the three nucleotides CGG will be repeated over and over. Fragile X syndrome is one of a number of neurodegenerative disorders caused by these triplet repeats of DNA. Other notable examples are Huntington disease, Friedreich’s ataxia, and myotonic dystrophy.

The number of these triplet repeats in the non-coding region of Fmr1 normally varies from individual to individual, but generally number between 5 and 50. From one generation to the next, though, the number of repeats can expand. Individuals with up to 200 of these trinucleotide repeats are considered asymptomatic carriers of a fragile X “premutation.” Though these carriers may not develop fragile X syndrome, there is a risk that their children will accumulate more repeats and will be at risk of fragile X syndrome.

In individuals with fragile X syndrome, the number of repeats is greater than 230, and sometimes the repeats can number in the thousands. Having too many of the triplet repeats interferes with the normal expression of the Fmr1 gene and can effectively silence Fmr1 transcription—keeping FMRP from being expressed and preventing the protein from performing its physiological functions.

But while the genetics of fragile X syndrome has been well characterized for several years, the mechanism by which Fmr1 mutations cause disease has not been as well understood. That’s because until recently, nobody has known what FMRP does in the body.

“There has been a lot of research in the last 10 years about what FMRP does,” says Vanderklish. “And in the last few years, there have been significant inroads.”

Today, it is generally understood that FMRP can act as a translation repressor—a protein that shuts off the expression of a gene at the very last step, just before it is translated from an mRNA transcript into a protein. But to understand how this actually causes fragile X syndrome, it is necessary to understand something about the shape and function of neurons.

Neuronal Spines Affected in Disease

While cells in the human body have a wide variety of shapes, neurons are perhaps the most varied. Many bear no resemblance to the ordered box-like structures visible under the microscope that several centuries ago caused Robert Hooke to coin the word “cell.”

Had Hooke been looking at neuronal cells instead of cork cells, he might have come up with a very different way of describing them. Neurons look more like Texas congressional districts gone wild—long and stretched-out, often covering much more ground than an ordinary cell. They do have a recognizable cell body, or soma, which contains the nucleus, but they also have incredibly elongated branching tendrils called dendrites. In some neurons, these dendritic “processes” extend for hundreds of times the length of the cell body and are so highly branched that they make synaptic contact with thousands of other neurons in the brain.

Dendrites play a key role in the function of neurons for it is through these processes that neurons communicate with one another. All along dendrites are synapses, the sites of communication between neurons where signals are communicated from one neuron to another via chemicals called neurotransmitters that are released across a synaptic cleft. Most synapses that use the amino acid glutamate as a neurotransmitter are characterized by the presence of a very small protrusion on the dendritic membrane called a dendritic “spine”. These are about one micron long (one millionth of a meter).

It is in this spiny part of neuronal cells that many of the processes believed to be essential for learning, memory and other higher brain functions take place. The branching, spiny architecture of dendrites is what allows a single neuron to receive up to tens of thousands inputs from other neurons—the communication backbone that underlies every mental process that a person has.

In the case of fragile X syndrome, dendritic spines are perturbed and show more soft underbelly than backbone. Under a microscope, the dendritic spines of people with fragile X syndrome are longer and appear more immature. They look more like those in a developing infant. This is a common observation in other forms of mental retardation as well.

One of the leading theories of the disease is that this altered shape may be affecting what is known as synaptic plasticity—a process whereby dendritic spines undergo subtle changes in shape and sensitivity to neurotransmitter in response to synaptic activity. Altered synaptic plasticity may be one of the most fundamental problems that causes mental retardation.

Would That They Were Plastic

Synaptic plasticity is an important process that takes place in neurons throughout life where activity at synapses triggers long-lasting changes in synaptic strength—called long-term depression and long-term potentiation. These changes are important during brain development, and for normal learning and memory throughout life, says Vanderklish, because they provide a basis for the long-term storage of information.

Based on the strength of neuronal signaling and the history of activity at a synapse, the junctions will change the shape of dendritic spines and their functional properties. One of the characteristics that these synaptic changes share with memory formation is that they require de novo protein synthesis for their consolidation. New proteins are needed rapidly. Generally speaking, they must be synthesized within 15 minutes or so of the induction of plasticity.

That presents a complicated engineering problem for neurons: How can they get the right cocktail of proteins to the correct dendritic spine in such a short period of time? Given that neurons may have as many as 10,000 synapses, how can one neuron express, assemble, package, and transport the diverse sets of enzymes and biological molecules needed hundreds of microns from the neuron’s soma to its most far-flung dendrites within a few minutes of the correct signal being received at any one dendritic spine?

This is a tall order for a cell, says Vanderklish, and for neurons to accomplish this, they must engage in a certain amount of premeditation—neurons package ready-made kits, called mRNA granules, and preship them to their far-flung dendrites where they might be needed. That way everything is already in place when the signal arrives.

These mRNA granules are like big cargo ships of protein and nucleic acid that contain the mRNA genetic transcripts and most of what is needed to make the proteins that consolidate synaptic plasticity. These include ribosomes, mRNAs, translation factors and the myriad other proteins and biological molecules required to make the final protein. The granules also include proteins that help to package mRNAs, motor proteins that help transport them to the end of the spines, and binding proteins to keep them in place once they get there.

In a sense, the neuron is loading a gun, cocking it, pointing it, and keeping a finger on the trigger until just that time when it is needed to fire.

Importantly, a further control is needed here—a safety to ensure that the genetic material in the granule is kept in an inactive form until it is truly needed. FMRP is hypothesized to be just such a safety. It is what is known as a “translation repressor.” It prevents mRNAs that are packaged into granules and transported to dendritic spines from becoming expressed too soon. It helps the granules stay in a silent state until the right signals arrive.

Loss of FMRP and Its Consequences

In fragile X syndrome, there is not enough of the FMRP to prevent translation of mRNA, and the inappropriate expression of proteins in response to synaptic activity is what researchers propose leads to mental retardation and other problems.

In mouse models that are lacking FMRP protein, Vanderklish and his colleagues have observed fewer of these granules. The granules have also been observed to be reorganized from the normal tight, spherical, and densely packed aggregates to less densely packed and unfurled ones, resembling something like beads on a string under an electron microscope. It is believed that when de novo protein synthesis is triggered by synaptic activity, granules are disassembled into clusters of ribosomes that are actively translating mRNAs. In fragile X syndrome, there is no FMRP around to limit this process so the amount of granules is reduced.

What effect does this have on the neurons? That has been a big question for the last 10 years or so, says Vanderklish. Now there is growing evidence that the loss of FMRP and control of protein expression at dendritic spines ultimately has an effect on synaptic plasticity. It seems to cause the immature shape of these spines that is characteristic of the syndrome. In the adult brain, this dendritic spine shape may be what allows a neuron to express a particular form of long-term depression, one that is enhanced in Fmr1 knockout mice.

But FMRP is not the only culprit, and that was really the subject of the poster and paper that Vanderklish and his colleagues recently presented. They and others have come to suspect there is another significant player in this process—a type of transmembrane receptor protein found at the surface of these dendritic spines called the metabotropic glutamate receptor (or “mGluR”) that triggers the form of long-term depression that is enhanced in Fmr1 knockout mice.

In recent years, a number of scientists, particularly Mark Bear of MIT, and Steve Warren of Emory University have begun to theorize that the activity of this metabotropic glutamate receptor is a major cause of the neuroanatomical and other anomalies associated with fragile X syndrome because it seems to work against FMRP by stimulating translation of mRNA present in FMRP-containing granules and inducing synaptic plasticity. It is thought that the loss of FMRP leads to enhanced translation and, therefore, an exaggeration of the functional and structural plasticity of synapses.

Support for this notion, says Vanderklish, can be seen in the results he and fellow Scripps Research investigator and Nobel laureate Gerald Edelman published a few years ago that showed when metabotropic glutamate receptors are stimulated, dendritic spines become elongated in cultured neurons, much as they do in the brains of people with fragile X syndrome.

And in their recent paper, Vanderklish, Edelman, and their colleagues were able to restore mRNA granule levels in Fmr1 knockout mice by supplying them with a chemical antagonist that blocks the activity of the metabotropic glutamate receptors. Blocking these receptors shut down their activity, and this caused the levels of granules to rebound in the knockout mice.

They also demonstrated that stimulating metabotropic glutamate receptors activates several other molecules that are involved in translation. This provides an important piece of evidence to support the theory that the critical neuronal mechanisms disrupted in fragile X syndrome are linked to metabotropic glutamate receptor-induced translation.

“It all comes together and points to a regulatory defect of metabotropic glutamate receptor-linked translation at synapses,” says Vanderklish.

This is significant because the metabotropic glutamate receptors might make an attractive target for designing therapies to treat fragile X syndrome. Such compounds have been shown to reverse many symptoms in the Fmr1 knockout mouse.

To read the article, “The fragile X mental retardation protein and group I metabotropic glutamate receptors regulate levels of mRNA granules in brain” by Armaz Aschrafi, Bruce A. Cunningham, Gerald M. Edelman, and Peter W. Vanderklish, please see the February 8, 2005 issue of the Proceedings of the National Academy of Sciences (2180-2185) or go to http://dx.doi.org/10.1073/pnas.0409803102

For a review of the mGluR theory of fragile X syndrome, see Bear et al. (2004), TINS 27(7): 370-377. To learn more about fragile X syndrome go to the FRAXA research foundation website.

Send comments to: jasonb@scripps.edu

Department of Neurobiology Research Associate Armaz Aschrafi (white shirt) discusses the details of his poster titled, "Altered mRNA granule composition and translation factor regulation in FMR1 knockout mice," which he prepared for the 2004 Society for Neurosciences meeting in San Diego.

Assistant Professor Peter Vanderklish standing in front of the same poster. The work was recently published in the journal PNAS. Photos by Jason Scorates Bardi.