Summary Of Fmrp Proposed Mechanism Of Action

FMRP seems to have a number of functions in the adult nervous system, and it is highly expressed in neurons where one of its functions is to transport and translate mRNAs38,39. Quite possibly the most important feature of FMRP is it's role in mRNP complexes. In the nucleus, FMRP can bind to mRNA, forming a messenger ribonucleoprotein, or mRNP. Via a nuclear export signal (NES), FMRP can leave the nucleus and attach to an anterograde motor protein, with its mRNA still bound. The mRNA and FMRP are then transported down the dendrite to spines or filopodia where one of three events can occur. First, the FMRP can go to filopodia and play a role in synaptogenesis. Second, the FMRP can go to spines, where it can regulate some of the protein synthesis that is required for their structure and maintenance. Third, FMRP can also repress protein synthesis at spines. In addition, FMRP in the dendrites could also return to the nucleus via its Nuclear Localization Signal (NLS)3,40,41. A summary of these possibilities is shown in Figure 30.2.

7. FMRP, mRNA, AND PROTEIN INTERACTIONS

FMRP is in a family of RNA-binding proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs). FMRP interacts with other proteins and forms part of a larger messenger ribonuclearprotein (mRNP) complex in the nucleus. FMRP normally associates with actively translating polyribosomes, and this association is affected by I304N missense mutation, which alters its ability to bind mRNA in vitro42-44. Possibly the most convincing evidence that mRNA binding plays a critical role in FXS comes from a single human FXS patient with severe symptoms, but no chromosomal constriction point45. This particular case of FXS was the result of a missense mutation, I304N, altering the folding of FMRP's second KH domain45. This KH domain likely plays an important role in the normal function of the FMRP protein, judging from the patient's symptoms. The KH domains are RNA-binding domains. The second KH domain in particular binds to a tertiary RNA structure called a kissing complex46. In addition to having two KH domains, FMRP has an RGG-type RNA-binding domain (an RGG box), and an amino terminus with a strong affinity for mRNA. In addition to FMRP binding to its own mRNA it also binds with varying specificity to an impressive estimate of 4% of all fetal mRNA's15. Further adding to the notion of the importance of FMRP's function as an RNA-binding protein are the extensive summaries of mouse mRNA FMRP is known to bind to. In 2001, a growing list of 432 mRNAs was available, and 251 of these have already been demonstrated to show abnormal polyribosomal profile in the absence of FMRP47. During development, polyribosomal aggregates increase in response during experience-dependant synaptogenesis in the spines, suggesting their importance in normal development.

Figure 30.2. Summary of Putative FMRP Actions in Dendrites and the Nucleus. As depicted in the figure, FMRP can associate with mRNA and other proteins in the nucleus to form mRNP. (1) mRNP is transported out of the nucleus into the cytoplasm. (2) where the FMRP/mRNA complex can affect translation through its associations with actively translating polyribosomes. (3) The FMRP/mRNA complex can also associate with motor proteins such as kinesin, and be transported down the dendrites to dendritic spines. (4) Near the synapse, FMRP is thought to regulate the translation of mRNA and respond to synaptic messages, including mGluR signaling. (5) In turn, mGluR5 signaling can also cause the production of FMRP at the synapse. (6) FMRP can also enter the nucleus, as it contains a NLS (figure courtesy of Colleen Webber).

Figure 30.2. Summary of Putative FMRP Actions in Dendrites and the Nucleus. As depicted in the figure, FMRP can associate with mRNA and other proteins in the nucleus to form mRNP. (1) mRNP is transported out of the nucleus into the cytoplasm. (2) where the FMRP/mRNA complex can affect translation through its associations with actively translating polyribosomes. (3) The FMRP/mRNA complex can also associate with motor proteins such as kinesin, and be transported down the dendrites to dendritic spines. (4) Near the synapse, FMRP is thought to regulate the translation of mRNA and respond to synaptic messages, including mGluR signaling. (5) In turn, mGluR5 signaling can also cause the production of FMRP at the synapse. (6) FMRP can also enter the nucleus, as it contains a NLS (figure courtesy of Colleen Webber).

FMRP expression can also be modulated by synaptic activity, and it is rapidly synthesized in response to the addition of glutamate and group 1 metabotropic glutamate receptor agonists48. Thus, this also suggests a role for FMRP in synapse formation and development, and it has been proposed that FMRP may be linked to dendritic regression48. FMRP can also bind to BC1, a dendritic nontranslatable brain mRNA, and the human analog BC200. The binding of BC1 mRNA is strong and takes place at the N-terminus of FMRP. This is a novel binding motif as it leaves the other three RNA-binding domains of the FMRP protein open. This may explain how FMRP regulates the translation of specific mRNAs at the synapse when FMRP is bound to BC1 RNA49. In this case, FMRP, in concert with BC1, can then repress the translation of specific mRNAs such as microtubule-associated protein 1B (MAP1b). In normal brain development, active synaptogenesis requires the decline of MAP1b, and in the FXS neuron, this protein may not be negatively regulated.

Further evidence that FMRP is linked to the cytoskeleton comes from FMRP's involvement in the murine Rac 1 pathway50'51. This pathway is involved in actin remodeling and is altered in fibroblasts that either lack FMRP, or have a point mutation involving the KH1 or KH2 RNA-binding domain. The MAP1b and the

Rac1 pathways could therefore be an important link to the control of the cytoskeleton during development, and a link between FMRP and the abnormal spine phenotype seen in men and mice missing this protein (i.e., see Figure 30.1).

FMRP has also been suggested to be a general repressor of translation, based on the evidence that the FMRP 1304N mutation fails to suppress translation in vivo51. Indeed, FMRP seems to be capable of inhibiting the formation of 80S ribosomal complexes52.

At the synapse, mRNA transport is important for transmission of signals. While it is difficult to identify the location of mRNA in the cell with many techniques, the development of "Antibody Positioned RNA Amplification" has shown for the first time that a loss of FMRP not only reduces certain mRNA's in vivo, but also results in a loss of their ability to localize to the dendrites53.

FMRP also has a role in the nucleus where it is associated with both a functional NES, and an NLS54. FMRP has also been shown to bind to single-stranded and double-stranded DNA in vitro, binding to double stranded DNA with a lower affinity.

As with most other proteins FMRP also has a host of interacting proteins that have been isolated and characterized. FXR1 and FXR2 are protein homologs of FMRP. FXR1 shares an approximate 90% identity with FMR1, both having almost identical KH domains55. FXR2 is very similar to FMRP, sharing 60% of their identities, also containing 2 KH domains, and abilities to bind to mRNA. FMR1, FXR1, and FXR2 can all form heteromers with each other, and all can form homomers, as well. They all interact tightly in the cytoplasm and nucleus56.

Two homologous cytoplasmic FMRP-interacting proteins, CYFIP1 and 2, also interact with FMRP, and CYFIP2 interacts with FXR1 and FXR2. Both are found localized at the synapses51. Although Drosophila only has dFMRP, lacking the orthologs for FXR1 and FXR2, there is a change in neural connectivity in this animal model. In the Drosophila model, Schenck et al. showed that expression of CYFIP, the Drosophila ortholog of CYFIP 1 and 2, was specific to the nervous system, and more importantly it links FMRP to the Rac1 GTPase pathway51. This essentially couples signal-dependant cytoskeleton remodeling and translation. Perhaps the most exciting link to FMRP's role in translation comes from recent electrophysiological data.

8. SYNAPTIC ELECTROPHYSIOLOGY AND THE mGLUR THEORY OF FXS

The loss of FMRP has been linked to several functional deficits in synaptic physiology. Alterations in synaptic plasticity have been observed in cortex57, the anterior cingulate cortex, lateral amygdala58, and hippocampus59. These alterations in synaptic physiology are not universal, and NMDA receptor-dependent forms of synaptic plasticity in the hippocampus are not altered in animals that lack FMRP18,59,60. Synaptic plasticity that involves group 1 metabotropic glutamate receptor activation is most dramatically affected by the loss of FMRP. Surprisingly, this does not produce a deficit, and instead mGluR-dependant LTD is significantly increased in the CA1 region of the hippocampus of FMR1 knockout mice. Moreover, this has been shown using two different methods for inducing mGluR LTD in vitro, while NMDAR-dependant LTD was found to be normal in these same experiments59.

With over 400 mRNA-binding partners of FMRP, and only a subtle behavioral phenotype for FMR1 knockout mouse models, identifying the main problems of these mice is a daunting task. If mGluR-dependant LTD is increased, then the loss of FMRP seems to result in an increase in the activity of the mGluR signaling pathway. Keeping in mind that mGluR requires rapid translation of pre-existing mRNA, and the fact that FMRP has been implicated in repressing translation, it was thought that FMRP could function to inhibit further protein synthesis caused by mGluR activation. Conversely, a lack of FMRP, as in FXS, would then theoretically lead to exaggerated mGluR signaling.

In addition to the electrophysiological data, it was pointed out that a number of other observations also support a role for altered mGluR signaling in FXS61. First, prepulse inhibition of an auditory startle reflex is normally decreased in mGluR1 and mGluR5 knockout mice, while it is increased in the FMR1 knockout mouse. Second, FXS is often associated with loose bowels, and mGluR5 is involved in the innervation of the ileum and mGluR agonists increase intestinal motility. Third, FXS is also characterized by a hypersensitivity to tactile stimulation, while mGluR5 receptors are involved in nocioception. In addition, there are a number of other anecdotal findings that give credence to the mGluR hypothesis for FXS61.

In summary, the mGluR theory predicts that the symptoms of FXS can be alleviated with specific mGluR antagonists. Theoretically, FMRP regulates a small portion of mRNAs, leaving other mRNAs to compete for resources for translation. Taking away FMRP upsets this delicate balance, and the potential treatment would involve restoring this balance with mGluR antagonists. Of course if the concentration of mGluR antagonists administered is too strong, the treatment could be equivocal to the disease itself as an imbalance would again be induced. For these reasons, and the fact that mGluR1 antagonists can disrupt cerebellar functioning, and cause ataxia, the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) has been more of a focus in current research. The effects of blocking mGluR5 are not likely to be beneficial however, as while mGluR5 knockout mice show decreased anxiety 62, they also show cognitive deficits63.

The selective mGluR5 antagonist MTEP (3-[(2-methyl-1,3-thiazol-4-yl) ethynyl]-pyridine) also seems to have some antidepressant effect on rats and mice64. With so many studies on FXS focusing on the relation between a few proteins, the attraction of the mGluR theory is that it gives a link between a single molecular mechanism and a number of behavioral and cognitive symptoms. In addition, the mGluR theory links one mechanism to the misregulation of many proteins. Although it is just a theory, it has already inspired some exciting discoveries, some of which are outlined below.

9. REPAIRING THE FRAGILE SYNAPSE 9.1. MPEP and Other mGluR Antagonists

MPEP and other mGluR antagonists seem to be effective in reversing some of the deficits observed in mGluR5 knockout mice. Administration of MPEP resulted in reversals for both the increased susceptibility to audiogenic seizures, and the decrease in open field activity (a measure of anxiety) normally common to these knockout animals65-67. The knockout mice developed a tolerance to MPEP, but this could be overcome by using higher doses.

Lithium chloride has long been in use in human patients for the treatment of mania, after being approved by the FDA in the 1970s. There are currently clinical trials ongoing to test the efficacy of lithium in FXS patients, and some evidence exists that there may be some benefits stabilizing mood swings in FXS patients19. The potential benefits of lithium have been brought to light by a research group that has looked at the effects of LiCl and MPEP in the Drosophila model of FXS. Both treatments restored some phenotypes of the animals missing dFMRP . Only two mGluRs are present in the Drosophila genome, DmGluRA and DmGluRB, more closely resembling vertebrate group 2 mGluRs. Possibly because of its effects on these mGluRs LiCl seemingly restored memory for courtship behavior in knockout flies, along with mushroom body deficits caused by the lack of dFMRP. Lithium is linked to the mGluR pathway through it s effects on CREB binding and inositol triphosphate receptor mediated calcium release. This has been outlined previously in relation to mGluR signalling in a study by McBride and colleagues65,68,70.

9.3. Gene Therapy

Gene therapy has the potential to cure FXS by simply restoring FMRP production. Admittedly, there are a number of obstacles to overcome, including finding the best structure of the gene, expressing this in cells, and the packaging and delivery of the gene. The use of an adenovirus has been suggested as one method of delivery, and it is hoped that the FMR1 knockout mouse will be an preclinical test of this therapy. The potential for the success of gene theraphy is illustrated in one large study, which has had success in both phase 1 and phase 2 clinical trials. Reduced lung function is the fatal symptom of cystic fibrosis, and using adenoviral infection as a method of delivery can improve human lung function in these patients71. Gene therapy does share some of the same problems as the pharmacological treatments however, as the ultimate goal remains to restore a delicate balance in the biochemical pathways involved with FMRP regulation. It remains to be determined how delicate the balance actually is, however, as male premutation carriers actually have elevated levels of FMRP and can exhibit symptoms associated with the premutation, FXTAS, later in life.

9.4. Other Options

FXS in humans is caused by hypermethylation and the recruitment of histone deacetylases which cause chromosome condensation that effectively shuts off this gene. Stopping this sequence of events would help greater than 95% of FXS patients who show a lack of FMRP as most individuals have a gene that can produce functional FMRP, rather than a point mutation. One study showed that in lymphoblastoid cells that contained 270-710 trinucleotide repeats (the full FXS mutation) FMRP production could still be seen13. In this case, the FMRP gene was reactivated modestly with the use of histone hyperacetylases. Using these in concert with DNA-demethylating agents, FMR1 mRNA levels were increased even more, often doubling the results seen with the use either one alone13. One recent review suggests that histone deacetylase inhibitors are potentially effective neuroprotective agents, and that further research in this area could help to treat diseases like FXS, leukemia, and various other cancers72. There is some evidence that Huntington's disease, Alzheimer's disease, amytrophic lateral sclerosis, and stroke could possibly benefit from this area of research, as well72.

Although there are complex biochemical changes in mice that lack FMRP, the knockout mouse phenotype deficiencies can be largely rescued by environmental enrichment, as well as increasing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit 1 (GluRl) levels. Paradoxically, environmental enrichment does not increase FMRP levels in wild type mice73. In some cases however, experience can change the expression and localization of FMRP74. Visual experience for example has been shown to regulate this phenomena in the visual cortex neurons74. Whisker stimulation can also cause neurons in the barrel cortex to translate FMRP (but this is stimulation dependant), and requires the activation of Group 1 mGluRs75.

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