Human YAC clones have been identified that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal to the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome (Verkerk, 1991).
Fragile X syndrome is associated with massive expansion of a CGG trinucleotide repeat within the FMR-1 gene and transcriptional silencing of the gene due to abnormal methylation. Partial cDNA sequence of the human FMR-1 has been reported. This study describes the isolation and characterization of cDNA clones encoding the murine homolog, fmr-1, which exhibits marked sequence identity with the human gene, including the conservation of the CGG repeat. A conserved ATG downstream of the CGG repeat in human and mouse and an in-frame stop codon in other human 5' cDNA sequences demarcate the FMR-1 coding region and confine the CGG repeat to the 5' untranslated region. Evidence is presented for alternative splicing of the FMR-1 gene in mouse and human brain; one of these splicing events alters the FMR-1 reading frame, predicting isoforms with novel carboxy termini (Ashley, 1993b).
Monoclonal antibodies specific for the FMR-1 protein have been raised. They detect 4-5 protein bands that appear identical in cells of normal males and of males carrying a premutation, but are absent in affected males with a full mutation. Immunohistochemistry shows a cytoplasmic localization of FMR-1. The highest levels are observed in neurons, while glial cells contain very low levels. In epithelial tissues, levels of FMR-1 are higher in dividing layers. In adult testis, FMR-1 is detected only in spermatogonia. FMR-1 is not detected in dermis and cardiac muscle except under pathological conditions (Devys, 1993).
Lack of expression of the fragile X mental retardation protein (FMRP) results in mental retardation and macroorchidism, seen as the major pathological symptoms in fragile X patients. FMRP is a cytoplasmic RNA-binding protein that cosediments with the 60S ribosomal subunit. Recently, two proteins homologous to FMRP were discovered: Fmr11 and Fmr12. These novel proteins interact with FMRP and with each other and they are also associated with the 60S ribosomal subunit. The expression pattern of the three proteins in brain and testis has been studied by immunohistochemistry. In adult brain, FMR1, Fmr11 and Fmr12 proteins are coexpressed in the cytoplasm of specific differentiated neurons only. However, a different expression pattern is observed in fetal brain as well as in adult and fetal testis, suggesting independent functions for the three proteins in those tissues during embryonic development and adult life (Tamanini, 1997).
FMRP, whose lack of expression causes the X-linked fragile X syndrome, is a modular RNA binding protein thought to be involved in posttranslational regulation. The structure in solution of the N-terminal domain of FMRP (NDF), a functionally important region involved in multiple interactions, is now know to consist of a composite fold comprising two repeats of a Tudor motif followed by a short alpha helix. The interactions between the three structural elements are essential for the stability of the NDF fold. Although structurally similar, the two repeats have different dynamic and functional properties. The second, more flexible repeat is responsible for interacting both with methylated lysine and with 82-FIP, one of the FMRP nuclear partners. NDF contains a 3D nucleolar localization signal, since destabilization of its fold leads to altered nucleolar localization of FMRP. It is suggested that the NDF composite fold determines an allosteric mechanism that regulates the FMRP functions.
Fragile X syndrome is the result of transcriptional suppression of the gene FMR1 as a result of a trinucleotide repeat expansion mutation. The normal function of the FMR1 protein (FMRP) and the mechanism by which its absence leads to mental retardation are unknown. Ribonucleoprotein particle (RNP) domains have been identified within FMRP, and RNA has been shown to bind in stoichiometric ratios, which suggests that there are two RNA binding sites per FMRP molecule. FMRP is able to bind to its own message with high affinity (dissociation constant = 5.7 nM) and interact with approximately 4 percent of human fetal brain messages. The absence of the normal interaction of FMRP with a subset of RNA molecules might result in the pleiotropic phenotype associated with fragile X syndrome (Ashley, 1993a).
FMR1 contains two types of sequence motifs recently found in RNA-binding proteins: an RGG box and two heterogeneous nuclear RNP K homology domains. FMR1 binds RNA in vitro. Using antibodies to FMR1, its expression is detected in divergent organisms and in cells of unaffected humans, but fragile X-affected patients express little or no FMR1. These findings demonstrate that FMR1 expression is directly correlated with the fragile X syndrome and suggest that anti-FMR1 antibodies will be important for diagnosis of fragile X syndrome. Furthermore, the RNA binding activity of FMR1 opens the way to understanding the function of FMR1 (Siomi, 1993).
Fmrp has been found to be a nucleocytoplasmic RNA-binding protein that contains both KH domains and RGG boxes; it associates with polyribosomes as a ribonucleoprotein particle. RNA binding has previously been demonstrated with in vitro-translated Fmrp; however, it remains uncertain whether the selective RNA binding observed is an intrinsic property of Fmrp or requires an associated protein(s). Here, baculovirus-expressed and affinity-purified FLAG-tagged murine Fmrp was shown to bind directly to both ribonucleotide homopolymers and human brain mRNA. FLAG-Fmrp exhibits selectivity for binding poly(G) > poly(U) >> poly(C) or poly(A). Moreover, purified FLAG-Fmrp binds to only a subset of brain mRNA, including the 3' untranslated regions of myelin basic protein message and its own message. Recombinant isoform 4, lacking the RGG boxes but maintaining both KH domains, was also purified and was found to only weakly interact with RNA. FLAG-purified I304N Fmrp, harboring the mutation of severe fragile X syndrome, demonstrates RNA binding. These data demonstrate the intrinsic property of Fmrp to selectively bind RNA and show FLAG-Fmrp as a suitable reagent for structural characterization and identification of cognate RNA ligands (Brown, 1998).
The identification and characterization are reported of a specific and high affinity binding site for FMRP in the RGG-coding region of its own mRNA. This site contains a purine quartet motif that is essential for FMRP binding and can be substituted by a heterologous quartet-forming motif. The specific binding of FMRP to its target site was confirmed further in a reticulocyte lysate through its ability to repress translation of a reporter gene harboring the RNA target site in the 5'-untranslated region. These data address interesting questions concerning the role of FMRP in the post-transcriptional control of its own gene and possibly other target genes (Schaeffer, 2001).
Since FMRP is an RNA-binding protein that associates with polyribosomes, it had been proposed to function as a regulator of gene expression at the post-transcriptional level. FMRP strongly inhibits translation of various mRNAs at nanomolar concentrations in both rabbit reticulocyte lysate and microinjected Xenopus laevis oocytes. This effect is specific for FMRP, since other proteins with similar RNA-binding domains, including the autosomal homologs of FMRP, Fmr11 and Fmr12, fail to suppress translation in the same concentration range. Strikingly, a disease-causing Ile-->Asn substitution at amino acid position 304 (I304N) renders FMRP incapable of interfering with translation in both test systems. Initial studies addressing the underlying mechanism of inhibition suggest that FMRP inhibits the assembly of 80S ribosomes on the target mRNAs. The failure of FMRP I304N to suppress translation is not due to its reduced affinity for mRNA or its interacting proteins Fmr11 and Fmr12. Instead, the I304N point mutation severely impairs homo-oligomerization of FMRP. These data support the notion that inhibition of translation may be a function of FMRP in vivo. It is further suggested that the failure of FMRP to oligomerize, caused by the I304N mutation, may contribute to the pathophysiological events leading to fragile X syndrome (Laggerbauer, 2001).
Microarray analysis of FMRP-associated brain mRNAs is reported in this study. mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. This approach has identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some of these mRNAs to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. It is concluded that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and candidate genes relevant to this phenotype were identified (Brown, 2001).
FMRP is a selective RNA-binding protein associated with polyribosomes. Purified recombinant FMRP causes a dose-dependent translational inhibition of brain poly(A) RNA in rabbit reticulocyte lysate without accelerated mRNA degradation. FMRP interacts with other messenger ribonucleoproteins and pre-exposure of FMRP to mRNA significantly increases the potency of FMRP as a translation inhibitor. Translation suppression by FMRP is reversed in a trans-acting manner by the 3'-untranslated portion of the Fmr1 message, which binds FMRP, suggesting that FMRP inhibits translation via interacting with mRNA. Consistently FMRP suppresses translation of the parathyroid hormone transcript, which binds FMRP, but not the beta-globin transcript, which does not bind FMRP. Moreover, removing the FMRP-binding site on a translation template abolishes the inhibitory effect of FMRP. Taken together, these results support the hypothesis that FMRP inhibits translation via interactions with the translation template (Li, 2001).
The fragile X syndrome results from amplification of the CGG repeat found in the FMR-1 gene. This CGG repeat shows length variation in normal individuals and is increased significantly in both carriers and patients; it is located 250 base pairs distal to a CpG island that is hypermethylated in fragile X patients. The methylation probably results in downregulation of FMR-1 gene expression. This study investigates the nature and function of the protein encoded by the FMR-1 gene using polyclonal antibodies raised against the predicted amino-acid sequences. Four different protein products, possibly resulting from alternative splicing, have been identified by immunoblotting in lymphoblastoid cell lines of healthy individuals. All these proteins were missing in cell lines from patients not expressing FMR-1 messenger RNA. The intracellular localization of the FMR-1 gene products was investigated by transient expression in COS-1 cells and found to be cytoplasmic. Localization was also predominantly cytoplasmic in the epithelium of the oesophagus, but in some cells was obviously nuclear (Verheij, 1993).
To gain insight into FMRP function, immunolocalization analysis of FMRP truncation and fusion constructs has been performed; this reveals a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggest that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP (Eberhart, 1996).
Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. Metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. mRNA has been identified for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. This mRNA associates with translational complexes in synaptoneurosomes within 1-2 min after mGluR1 stimulation of this preparation: increased expression of FMRP is observed after mGluR1 stimulation. In addition, FMRP is associated with polyribosomal complexes in these fractions. In vivo, FMRP immunoreactivity is observed in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. It is suggested that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections (Weiler, 1997).
A detailed cellular localization study of FMRP has been carried out using both biochemical and immunocytochemical approaches. FMRP is highly expressed in neurons but not glia throughout the rat brain, as detected by light microscopy. Although certain structures, such as hippocampus, reveal a strong signal, the regional variation in staining intensity appears to be related to neuron size and density. In human cell lines and mouse brain, FMRP co-fractionates primarily with polysomes and rough endoplasmic reticulum. Ultrastructural studies in rat brain have revealed high levels of FMRP immunoreactivity in neuronal perikarya, where FMRP is concentrated in regions rich in ribosomes, particularly near or between rough endoplasmic reticulum cisternae. Immunogold studies has provides evidence of nucleocytoplasmic shuttling of FMRP, which was localized in neuronal nucleoplasm and within nuclear pores. Moreover, labeling is observed in large- and small-caliber dendrites, in dendritic branch points, at the origins of spine necks, and in spine heads, all known locations of neuronal polysomes. Dendritic localization, which was confirmed by co-fractionation of FMRP with synaptosomal ribosomes, suggests a possible role of FMRP in the translation of proteins involved in dendritic structure or function and relevant for the mental retardation occurring in fragile X syndrome (Feng, 1997b).
Loss of fragile X mental retardation protein (FMRP) function causes the fragile X mental retardation syndrome. FMRP harbors three RNA binding domains, associates with polysomes, and is thought to regulate mRNA translation and/or localization, but the RNAs to which it binds are unknown. RNA selection was used to demonstrate that the FMRP RGG box binds intramolecular G quartets. These data allowed the identification of mRNAs encoding proteins involved in synaptic or developmental neurobiology that harbor FMRP binding elements. The majority of these mRNAs have an altered polysome association in fragile X patient cells. These data demonstrate that G quartets serve as physiologically relevant targets for FMRP and identify mRNAs whose dysregulation may underlie human mental retardation (Darnell, 2001).
The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. Antibody-positioned RNA amplification (APRA) has been developed to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP, using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, it has been observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP (Miyashiro, 2003).
The Fragile X syndrome, which results from the absence of functional FMRP protein, is the most common heritable form of mental retardation. FMRP acts as a translational repressor of specific mRNAs at synapses. Interestingly, FMRP associates not only with these target mRNAs, but also with the dendritic, non-translatable RNA BC1. Blocking of BC1 inhibits the interaction of FMRP with its target mRNAs. Furthermore, BC1 binds directly to FMRP and can also associate, in the absence of any protein, with the mRNAs regulated by FMRP. This suggests a mechanism where BC1 could determine the specificity of FMRP function by linking the regulated mRNAs and FMRP. Thus, when FMRP is not present, loss of translational repression of specific mRNAs at synapses results in synaptic dysfunction phenotype of Fragile X patients (Zalfa, 2003).
Fragile X syndrome, the most common form of inherited mental retardation, is caused by absence of FMRP, an RNA-binding protein implicated in regulation of mRNA translation and/or transport. dFMR1, the Drosophila ortholog of FMRP, is genetically linked to the dRac1 GTPase, a key player in actin cytoskeleton remodeling. This study demonstrates that FMRP and the Rac1 pathway are connected in a model of murine fibroblasts. Rac1 activation induces relocalization of four FMRP partners to actin ring areas. Moreover, Rac1-induced actin remodeling is altered in fibroblasts lacking FMRP or carrying a point-mutation in the KH1 or in the KH2 RNA-binding domain. In absence of wild-type FMRP, it was found that phospho-ADF/Cofilin (P-Cofilin) level, a major mediator of Rac1 signaling, is lowered, whereas the level of protein phosphatase 2A catalytic subunit (PP2Ac), a P-Cofilin phosphatase, is increased. FMRP binds with high affinity to the 5'-UTR of pp2acbeta mRNA and is thus a likely negative regulator of its translation. The molecular mechanism unraveled here points to a role for FMRP in modulation of actin dynamics, which is a key process in morphogenesis of dendritic spines, synaptic structures abnormally developed in Fragile X syndrome patient's brain (Castets, 2005).
The FMR1 transcript is alternatively spliced and generates different splice variants coding for FMR1 proteins (FMRP) with a predicted molecular mass of 70-80 kDa. FMRP is widely expressed and localized in the cytoplasm. To study a possible interaction with other cellular components, FMRP was isolated and characterized under non-denaturing conditions. Under physiological salt conditions FMRP appears to have a molecular mass of > 600 kDa, indicating a binding to other cellular components. This interaction is disrupted in the presence of high salt concentrations. The dissociation conditions to free FMRP from the complex are similar to the dissociation of FMRP from RNA as shown before. The binding of FMRP from the complex is also disrupted by RNAse treatment. That the association of FMRP to a high molecular weight complex possibly occurs via RNA, is further supported by the observation that the binding of FMRP, containing an lle304Asn substitution, to the high molecular weight complex is reduced. An equal reduced binding of mutated FMRP to RNA in vitro was observed before under the same conditions. The reduced binding of FMRP with the lle304Asn substitution further indicates that the interaction to the complex indeed occurs via FMRP and not via other RNA binding proteins. In a reconstitution experiment where the low molecular mass FMRP (70-80 kDa) was mixed with a reticulocyte lysate (enriched in ribosomes) it was shown that FMRP can associate to ribosomes and that this binding most likely occurs via RNA (Tamanini, 1996).
Fragile-X mental retardation is caused by loss of function of a single gene encoding the Fragile-X mental retardation protein, FMRP, an RNA-binding protein that harbors two KH-type and one RGG-type RNA-binding domains. Previous studies identified intramolecular G-quartet RNAs as high-affinity targets for the RGG box, but the relationship of RNA binding to FMRP function and mental retardation remains unclear. One severely affected patient harbors a missense mutation (I304N) within the second KH domain (KH2), and some evidence suggests this domain may be involved in the proposed role of FMRP in translational regulation. The RNA target for the KH2 domain has been identied as a sequence-specific element within a complex tertiary structure termed the FMRP kissing complex (kc). This study demonstrates that the association of FMRP with brain polyribosomes is abrogated by competition with the FMRP kissing complex RNA, but not by high-affinity G-quartet RNAs. It is concluded that mental retardation associated with the I304N mutation, and likely the Fragile-X syndrome more generally, may relate to a crucial role for RNAs harboring the kissing complex motif as targets for FMRP translational regulation (Darnell, 2005).
Prior structural studies have demonstrated that KH domains bind to single-stranded regions of RNA, and these may be presented in the context of stem-loop structure. To further explore the structure of the kc RNA and its relationship to KH2 binding, positions in the RNA accessible to chemical modification were examined, using reagents that modify unpaired nucleotides and detecting modifications by stops in primer extension. Since KH2:kc RNA binding requires the presence of Mg+2, results obtained in the presence of Mg+2 versus EDTA were compared. DMS, CMCT, and kethoxal (which modify unpaired N3-C and N1-A, N3-U and N1-G, and N1-G and N2-G, respectively) were examined for chemical modification. It was found that kc RNA was modified in EDTA, but was protected in Mg+2 in several regions. The data suggest the presence of an unanticipated 4-bp duplex forming between the two loops (a conserved 5'-C48UGG51-3' in the 3' loop and a 5'-C11CAG14-3' present in the fixed sequence of the 5' loop). In addition, an A20-U45 pair previously found to covary with G20-C45 was also protected in the presence of Mg+2, suggesting formation of a Watson-Crick base pair. These experiments also provide evidence that the stem of the 5' stem-loop predicted by mfold (5'-UUCC-3' with 5'-GGAG-3') folds in a Mg+2-dependent manner. The stem of the 3' stem-loop predicted by mfold is base-paired in a Mg+2-independent manner. This finding, that there is a 4-bp interaction between the loops and that the R20 and Y45 nucleotides base pair with each other, provides strong evidence that the two loops form a stable loop-loop pseudoknot, or kissing complex (Darnell, 2005).
The mechanism by which FMRP might interact with polyribosomes has not been well understood. FMRP has been reported to interact with a number of RNA-binding proteins, including FXR1P/FXR2P, YB-1, Staufen, and nucleolin, consistent with data suggesting that FMRP may be part of large mRNPs, some of which may be associated with polyribosomes. In addition, FMRP copurifies with the RISC complex in Drosophila and coimmunoprecipitates with the dAgo-1 homolog, a protein component of the RISC complex in mammalian cells in culture. These observations have suggested that FMRP might bind to polyribosomes indirectly, via protein-RNA networks, and/or RISC complex-targeting of miRNAs to mRNAs. For example, it has been suggested that FMRP might 'scan' mRNAs for G-quartet elements that then become associated with RISC complex proteins in a translationally regulated polyribosome complex. The current data indicate that this model needs revision. G-quartet RNAs may be involved in RNA binding, translational control, and even polyribosome association in the brain. However, G-quartets do not compete FMRP off polyribosomes, and deletion of the RGG-box does not affect FMRP polyribosome association, suggesting that G-quartets are not required for polyribosome association. Instead, the data suggest that direct association of FMRP with RNAs harboring kissing complex motifs fitting the consensus described in this study mediate FMRP association with brain polyribosomes (Darnell, 2005).
The association of FMRP with brain polyribosomes is specifically disrupted by competition with kissing complex RNAs fitting the consensus described here. Furthermore, the FMRP:kissing complex interaction is mediated by KH2 and is sensitive to the I304N mutation. Taken together, these findings indicate that these kissing complex RNAs may provide a crucial link between the association of FMRP in brain polyribosomes, its proposed role in regulating mRNA translation, and neurologic dysfunction in the Fragile-X syndrome. Redirecting the ongoing search for FMRP RNA targets to those RNAs harboring kissing complex motifs may be of importance in identifying RNA ligands central to the Fragile-X syndrome (Darnell, 2005).
Fragile X [fraX] syndrome is a common hereditary disorder associated with a fragile site marker at Xq27.3 that clinically presents as a form of mental retardation (MR). Postmortem investigation of 3 fraX positive males with mild to moderate MR did not document any gross neuropathological changes. Golgi analysis of neocortical dendritic spine morphology extended previous observations of immature, long, tortuous spines in one adult case of fraX to 2 new cases. Evidence for similar dendritic spine abnormalities was found, although Golgi analysis was less than optimal because of incomplete dendritic stain impregnation. Neocortical intra-layer cell density was also investigated in all 3 cases. Cresyl violet stained neurons were counted in 10 randomly selected fields in neocortical layers II-VI of cingulate and temporal association areas (Brodmann's areas 23 and 38). Neuron counts in fraX and control neocortex showed no significant differences. Thus, abnormal dendritic spine morphology with preservation of neuronal density appears to characterize the neocortex in individuals with this common form of mental retardation (Hinton, 1991).
A missense mutation in FMRP, I304N, has been found to result in an unusually severe phenotype. Normal FMRP associates with elongating polyribosomes via large mRNP particles. Despite normal expression and cytoplasmic mRNA association, the I304N FMRP is incorporated into abnormal mRNP particles that are not associated with polyribosomes. These data indicate that association of FMRP with polyribosomes must be functionally important and imply that the mechanism of the severe phenotype in the I304N patient lies in the sequestration of bound mRNAs in nontranslatable mRNP particles. In the absence of FMRP, these same mRNAs may be partially translated via alternative mRNPs, although perhaps abnormally localized or regulated, resulting in typical fragile X syndrome (Feng, 1997a).
Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. This study describes dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (Fmr1) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice are longer than those in wild-type mice and are often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite is greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning (Comery, 1997).
Qualitative examination of human brain autopsy material has shown that fragile-X patients exhibit abnormal dendritic spine lengths and shapes on parieto-occipital neocortical pyramidal cells. Dendritic spines on layer V pyramidal cells of human temporal and visual cortices stained using the Golgi-Kopsch method were investigated. Quantitative analysis of dendritic spine length, morphology, and number was carried out on patients with fragile-X syndrome and normal age-matched controls. Fragile-X patients exhibit significantly more long dendritic spines and fewer short dendritic spines than do control subjects in both temporal and visual cortical areas. Similarly, fragile-X patients exhibit significantly more dendritic spines with an immature morphology and fewer with a more mature type morphology in both cortical areas. In addition, fragile-X patients had a higher density of dendritic spines than did controls on distal segments of apical and basilar dendrites in both cortical areas. Long dendritic spines with immature morphologies and elevated spine numbers are characteristic of early development or a lack of sensory experience. The fact that these characteristics are found in fragile-X patients throughout multiple cortical areas may suggest a global failure of normal dendritic spine maturation and or pruning during development that persists throughout adulthood (Irwin, 2001).
Reports that patients and adult FMR1 knock-out mice have abnormally long dendritic spines of increased density suggested that Fragile X syndrome might involve abnormal spine development. Because spine length, density, and motility change dramatically in the first postnatal weeks, these properties were analyzed in mutant mice and littermate controls at 1, 2, and 4 weeks of age. To label neurons, a viral vector carrying the enhanced green fluorescent protein gene was injected into the barrel cortex. Layer V neurons were imaged on a two-photon laser scanning microscope in fixed tissue sections. Analysis of >16,000 spines showed clear developmental patterns. Between 1 and 4 weeks of age, spine density increases 2.5-fold, and mean spine length decreased by 17% in normal animals. Early during cortical synaptogenesis, pyramidal cells in mutant mice have longer spines than controls. At 1 week, spine length is 28% greater in mutants than in controls. At 2 weeks, this difference is 10%, and at 4 weeks only 3%. Similarly, spine density is 33% greater in mutants than in controls at 1 week of age. At 2 or 4 weeks of age, differences are not detectable. The spine abnormality is not detected in neocortical organotypic cultures. The transient nature of the spine abnormality in the intact animal suggests that FMRP might play a role in the normal process of dendritic spine growth in coordination with the experience-dependent development of cortical circuits (Nimchinsky, 2001).
Absence of functional FMRP causes Fragile X syndrome. Abnormalities in synaptic processes in the cerebral cortex and hippocampus contribute to cognitive deficits in Fragile X patients. So far, the potential roles of cerebellar deficits have not been investigated. This study demonstrates that both global and Purkinje cell-specific knockouts of Fmr1 show deficits in classical delay eyeblink conditioning in that the percentage of conditioned responses as well as their peak amplitude and peak velocity are reduced. Purkinje cells of these mice show elongated spines and enhanced LTD induction at the parallel fiber synapses that innervate these spines. Moreover, Fragile X patients display the same cerebellar deficits in eyeblink conditioning as the mutant mice. These data indicate that a lack of FMRP leads to cerebellar deficits at both the cellular and behavioral levels and raise the possibility that cerebellar dysfunctions can contribute to motor learning deficits in Fragile X patients (Koekkoek, 2005).
Fragile X syndrome, caused by a mutation in the Fmr1 gene, is characterized by mental retardation. Several studies reported the absence of long-term potentiation (LTP) at neocortical synapses in Fmr1 knockout (FMR1-KO) mice, but underlying cellular mechanisms are unknown. This study found that in the prefrontal cortex (PFC) of FMR1-KO mice, spike-timing-dependent LTP (tLTP) is not so much absent, but rather, the threshold for tLTP induction is increased. Calcium signaling in dendrites and spines is compromised. (1) Dendrites and spines more often fail to show calcium transients. (2) The activity of L-type calcium channels is absent in spines. tLTP could be restored by improving reliability and amplitude of calcium signaling by increasing neuronal activity. In FMR1-KO mice that were raised in enriched environments, tLTP was restored to WT levels. These results show that mechanisms for synaptic plasticity are in place in the FMR1-KO mouse PFC, but require stronger neuronal activity to be triggered (Meredith, 2007).
The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. This study reports a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs are deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity (Dictenberg, 2008).
The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) have been identified as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP--CYFIP interaction involves the domain of FMRP also mediating homo- and hetero-merization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts (Schenck, 2001).
Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently, mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Mammalian FMRP is show to interact in vitro with microRNAs and the components of the microRNA pathways including Dicer and the mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, it is shown that AGO1 is critical for FMRP function in neural development and synaptogenesis. These results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease (Jin, 2004).
As a step toward understanding the function of FMR1 and the determination of the potential for therapeutic approaches to fragile X syndrome, yeast artificial chromosome (YAC) transgenic mice were generated in order to determine whether the Fmr1 knockout mouse phenotype could be rescued. Several transgenic lines were generated that carried the entire FMR1 locus with extensive amounts of flanking sequence. The YAC transgene supports production of the human protein (FMRP) which was present at levels 10 to 15 times that of endogenous protein and was expressed in a cell- and tissue-specific manner. Macro-orchidism is absent in knockout mice carrying the YAC transgene indicating functional rescue by the human protein. Given the complex behavioral phenotype in fragile X patients and the mild phenotype previously reported for the Fmr1 knockout mouse, a more thorough evaluation of the Fmr1 knockout phenotype was performed using additional behavioral assays that had not previously been reported for this animal model. The mouse displays reduced anxiety-related responses with increased exploratory behavior. FMR1 YAC transgenic mice overexpressing the human protein produce opposing behavioral responses and additional abnormal behaviors were also observed. These findings have significant implications for gene therapy for fragile X syndrome since overexpression of the gene may harbor its own phenotype (Peier, 2000).
The fragile X mental retardation protein (FMRP) is an RNA-binding protein that controls translational efficiency and regulates synaptic plasticity. FMRP is involved in dopamine (DA) modulation of synaptic potentiation. AMPA glutamate receptor subtype 1 (GluR1) surface expression and phosphorylation in response to D1 receptor stimulation were reduced in cultured Fmr1−/− prefrontal cortex (PFC) neurons. Furthermore, D1 receptor signaling was impaired, accompanied by D1 receptor hyperphosphorylation at serine sites and subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in both PFC and striatum of Fmr1−/− mice. FMRP interacted with GRK2, and pharmacological inhibition of GRK2 rescued D1 receptor signaling in Fmr1−/− neurons. Finally, D1 receptor agonist partially rescued hyperactivity and enhanced the motor function of Fmr1−/− mice. This study has identified FMRP as a key messenger for DA modulation in the forebrain and may provide insights into the cellular and molecular mechanisms underlying fragile X syndrome (Wang, 2008).
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