Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure

Fragile X syndrome (FraX), caused by the loss-of-function of one gene (FMR1), is the most common inherited form of both mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an mRNA-binding translation regulator that mediates activity-dependent control of synaptic structure and function. To develop any FraX intervention strategy, it is essential to define when and where FMRP loss causes the manifestation of synaptic defects, and whether the reintroduction of FMRP can restore normal synapse properties. In the Drosophila FraX model, dFMRP loss causes neuromuscular junction (NMJ) synapse over-elaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of development-arrested satellite boutons, and altered neurotransmission. We used the Gene-Switch method to conditionally drive dFMRP expression to define the spatiotemporal requirements in synaptic mechanisms. Constitutive induction of targeted neuronal dFMRP at wild-type levels rescues all synaptic architectural defects in Drosophila Fmr1 (dfmr1)-null mutants, demonstrating a presynaptic requirement for synapse structuring. By contrast, presynaptic dFMRP expression does not ameliorate functional neurotransmission defects, indicating a postsynaptic dFMRP requirement. Strikingly, targeted early induction of dFMRP effects nearly complete rescue of synaptic structure defects, showing a primarily early-development role. In addition, acute dFMRP expression at maturity partially alleviates dfmr1-null defects, although rescue is not as complete as either early or constitutive dFMRP expression, showing a modest capacity for late-stage structural plasticity. We conclude that dFMRP predominantly acts early in synaptogenesis to modulate architecture, but that late dFMRP introduction at maturity can weakly compensate for early absence of dFMRP function.     

Dynamic translational and proteasomal regulation of fragile X mental retardation protein controls mGluR-dependent long-term depression

Genetic deletion of fragile X mental retardation protein (FMRP) has been shown to enhance mGluR-dependent long-term depression (LTD). Herein, we demonstrate that mGluR-LTD induces a transient, translation-dependent increase in FMRP that is rapidly degraded by the ubiquitin-proteasome pathway. Moreover, proteasome inhibitors abolished mGluR-LTD, and LTD was absent in mice that overexpress human FMRP. Neither translation nor proteasome inhibitors blocked the augmentation of mGluR-LTD in FMRP-deficient mice. In addition, mGluR-LTD is associated with rapid increases in the protein levels of FMRP target mRNAs in wild-type mice. Interestingly, the basal levels of these proteins were elevated and their synthesis was improperly regulated during mGluR-LTD in FMRP-deficient mice. Our findings indicate that hippocampal mGluR-LTD requires the rapid synthesis and degradation of FMRP and that mGluR-LTD triggers the synthesis of FMRP binding mRNAs. These findings indicate that the translation, ubiquitination, and proteolysis of FMRP functions as a dynamic regulatory system for controlling synaptic plasticity.     

The X chromosome and fragile X mental retardation

Fragile X syndrome represents the most common inherited cause of mental retardation. It is caused by a stretch of CGG repeats within the fragile X gene, which increases in length as it is transmitted from generation to generation. Once the repeat exceeds a threshold length, no protein is produced, resulting in the fragile X phenotype. Both X chromosome inactivation and inactivation of the FMR1 gene are the result of methylation. X inactivation occurs earlier than inactivation of the FMR1 gene. The instability to a full mutation is dependent on the sex of the transmitting parent and occurs only from mother to child. For most X-chromosomal diseases, female carriers do not express the phenotype. A clear exception is fragile X syndrome. It is clear that more than 50% of the neurons have to express the protein to ensure a normal phenotype in females. This means that a normal phenotype in female carriers of a full mutation is accompanied by a distortion of the normal distribution of X inactivation.     

Biology of the fragile X mental retardation protein, an RNA-binding protein.

The fragile X syndrome, an X-linked disease, is the most frequent cause of inherited mental retardation. The syndrome results from the absence of expression of the FMR1 gene (fragile mental retardation 1) owing to the expansion of a CGG trinucleotide repeat located in the 5' untranslated region of the gene and the subsequent methylation of its CpG island. The FMR1 gene product (FMRP) is a cytoplasmic protein that contains two KH domains and one RGG box, characteristics of RNA-binding proteins. FMRP is associated with mRNP complexes containing poly(A)+mRNA within actively translating polyribosomes and contains nuclear localization and export signals making it a putative transporter (chaperone) of mRNA from the nucleus to the cytoplasm. FMRP is the archetype of a novel family of cytoplasmic RNA-binding proteins that includes FXR1P and FXR2P. Both of these proteins are very similar in overall structure to FMRP and are also associated with cytoplasmic mRNPs. Members of the FMR family are widely expressed in mouse and human tissues, albeit at various levels, and seem to play a subtle choreography of expression. FMRP is most abundant in neurons and is absent in muscle. FXR1P is strongly expressed in muscle and low levels are detected in neurons. The complex expression patterns of the FMR1 gene family in different cells and tissues suggest that independent, however similar, functions for each of the three FMR-related proteins might be expected in the selection and metabolism of tissue-specific classes of mRNA. The molecular mechanisms altered in cells lacking FMRP still remain to be elucidated as well as the putative role(s) of FXR1P and FXR2P as compensatory molecules.     

Fragile X mental retardation syndrome: structure of the KH1-KH2 domains of fragile X mental retardation protein

Fragile X syndrome is the most common form of inherited mental retardation in humans, with an estimated prevalence of about 1 in 4000 males. Although several observations indicate that the absence of functional Fragile X Mental Retardation Protein (FMRP) is the underlying basis of Fragile X syndrome, the structure and function of FMRP are currently unknown. Here, we present an X-ray crystal structure of the tandem KH domains of human FMRP, which reveals the relative orientation of the KH1 and KH2 domains and the location of residue Ile304, whose mutation to Asn is associated with a particularly severe incidence of Fragile X syndrome. We show that the Ile304Asn mutation both perturbs the structure and destabilizes the protein.     

Drosophila fragile X mental retardation protein developmentally regulates activity-dependent axon pruning

Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes, as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both: a distinction that is vital for engineering intervention strategies. To address this crucial issue, we have used the Drosophila FraX model to investigate the developmental function of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and is required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement during late brain development.     

RNA and microRNAs in fragile X mental retardation

Fragile X syndrome is caused by the loss of an RNA-binding protein called FMRP (for fragile X mental retardation protein). FMRP seems to influence synaptic plasticity through its role in mRNA transport and translational regulation. Recent advances include the identification of mRNA ligands, FMRP-mediated mRNA transport and the neuronal consequence of FMRP deficiency. FMRP was also recently linked to the microRNA pathway. These advances provide mechanistic insight into this disorder, and into learning and memory in general.     

Alternative splicing modulates protein arginine methyltransferase-dependent methylation of fragile X syndrome mental retardation protein

The fragile X mental retardation protein (FMRP) is an RNA binding protein that is methylated by an endogenous methyltransferase in rabbit reticulocyte lysates. We mapped the region of methylation to the C-terminal arginine-glycine-rich residues encoded by FMR1 exon 15. We additionally demonstrated that mutation of R(544) to K reduced the endogenous methylation by more than 80%, while a comparable mutant R(546)-K reduced the endogenous methylation by 20%. These mutations had no effect on the subcellular distribution of FMRP, recapitulating previous results using the methyltransferase inhibitor adenosine-2',3'-dialdehyde. Using purified recombinant protein arginine methyltransferases (PRMTs), we showed that the C-terminal domain could be methylated by PRMT1, PRMT3, and PRMT4 in vitro and that both the R(544)-K mutant and the R(546)-K mutant were refractory toward these enzymes. We also report that truncating the N-terminal 12 residues encoded by FMR1 exon 15, which occurs naturally via alternative splicing, had no effect on FMRP methylation, demonstrating conclusively that phosphorylation of serine residue 500 (S(500)), one of the 12 residues, was not required for methylation. Nevertheless, truncating 13 additional amino acids, as occurs in the smallest alternatively spliced variant of FMR1 exon 15, reduced methylation by more than 85%. This suggests that differential expression and methylation of the FMRP exon 15 variants may be an important means of regulating target mRNA translation, which is consonant with recently demonstrated functional effects mediated by inhibiting FMRP methylation in cultured cells.     

Balanced X-autosomal translocation and mental retardation. Mapping mental retardation linked to X (excluding fragile X)

From personal observations and reported cases of translocation X-Autosome, a study of the breakpoint showed that Xp11 is more frequently associated to mental retardation. This finding is in agreement with linkage analysis in families with X-linked mental retardation non X-fra.     

Contribution of voltage-gated potassium channels to the regulation of apoptosis

Recent evidence points to the crucial involvement of voltage-gated potassium channels (Kv) in apoptotic volume decrease and in the regulation of apoptosis in several systems. We have recently described the presence of a Kv channel, Kv1.3, in the mitochondria of lymphocytes. Expression of the channel correlated with increased sensitivity to apoptotic stimuli. Mitochondrial Kv1.3 contributes to the apoptotic cascade in T lymphocytes by interacting with pro-apoptotic Bax resulting in alteration of mitochondrial functional parameters and ultimately, in cytochrome c release. The present review summarizes the current understanding of the function of Kv channels in apoptosis in several cell types as well as the role of mitochondrial Kv1.3 in the regulation of cell death in lymphocytes.     

The Drosophila fragile X mental retardation protein controls actin dynamics by directly regulating profilin in the brain

Loss of Fragile X mental retardation protein (FMRP) function causes the highly prevalent Fragile X syndrome [1 and 2]. Identifying targets for the RNA binding FMRP is a major challenge and an important goal of research into the pathology of the disease. Perturbations in neuronal development and circadian behavior are seen in Drosophila dfmr1 mutants. Here we show that regulation of the actin cytoskeleton is under dFMRP control. dFMRP binds the mRNA of the Drosophila profilin homolog and negatively regulates Profilin protein expression. An increase in Profilin mimics the phenotype of dfmr1 mutants. Conversely, decreasing Profilin levels suppresses dfmr1 phenotypes. These data place a new emphasis on actin misregulation as a major problem in fmr1 mutant neurons.     

Fragile X related protein 1 isoforms differentially modulate the affinity of fragile X mental retardation protein for G-quartet RNA structure

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of expression of the Fragile X Mental Retardation Protein (FMRP), an RNA binding protein with high specificity for G-quartet RNA structure. FMRP is involved in several steps of mRNA metabolism: nucleocytoplasmic trafficking, translational control and transport along dendrites in neurons. Fragile X Related Protein 1 (FXR1P), a homologue and interactor of FMRP, has been postulated to have a function similar to FMRP, leading to the hypothesis that it can compensate for the absence of FMRP in Fragile X patients. Here we analyze the ability of three isoforms of FXR1P, expressed in different tissues, to bind G-quartet RNA structure specifically. Only the longest FXR1P isoform was found to be able to bind specifically the G-quartet RNA, albeit with a lower affinity as compared to FMRP, whereas the other two isoforms negatively regulate the affinity of FMRP for G-quartet RNA. This result is important to decipher the molecular basis of fragile X syndrome, through the understanding of FMRP action in the context of its multimolecular complex in different tissues. In addition, we show that the action of FXR1P is synergistic rather than compensatory for FMRP function.     

Dynamic modification of dendritic cable properties and synaptic transmission by voltage-gated potassium channels

Computer simulations of a dendrite possessing voltage-sensitive potassium conductances were used to determine the effects of these conductances on synaptic transmission and on the propagation of synaptic signals within the dendritic tree. Potassium conductances had two principal effects on voltage transients generated by current injections or synaptic conductances. Locally (near the source of the transient), voltage-gated potassium channels produced a potassium shunt current that reduced the amplitude of voltage transients generated by depolarizing currents. This shunt current increased as the amplitude of the depolarizing transient increased and so acted to prevent large synaptic transients from reaching levels that would saturate due to a reduction in driving force. In the presence of rapidly activating potassium currents, excitatory synapses produced larger synaptic currents that were more linearly related to synaptic conductance, but these produced smaller voltage transients. The maximum amplitudes of the voltage transients were limited by the voltage sensitivity of the K⁺ conductance and the rate at which it could activate. Sufficiently rapid synaptic currents could outrun the K⁺ conductance and thus achieve high local peak amplitudes. These effects of K⁺ conductances were unrelated to whether they were located on dendrites or not, being related only to their proximity to the source of synaptic current. The second class of effects of K⁺ conductances depended on their alteration of the electrotonic structure of the postsynaptic cell and so were observed only when they were located on postsynaptic dendrites. Voltage-gated K⁺ conductances produced voltage-dependent electrotonic expansion of depolarized dendrites, which had the effect of isolating synaptic inputs on depolarized dendrites from events on the rest of the neuron. Thus, synapses on the same dendrite interacted destructively to a degree much greater than that expected from the classical driving force nonlinearity. Synapses located proximally to a depolarized dendritic region were less effected than those located distally, and the range of the nonlinear interaction between synapses was dependent on the kinetics of activation and deactivation of the conductance. When present in conjunction with rapidly activating dendritic sodium conductance, the potassium conductance sharpened the requirement for spatial and temporal coincidence to produce synaptic boosting by inward currents, and suppressed out-of-synchrony synaptic inputs.     

Characterization of dFMR1, a Drosophila melanogaster homolog of the fragile X mental retardation protein

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by loss of FMR1 gene activity due to either lack of expression or expression of a mutant form of the protein. In mammals, FMR1 is a member of a small protein family that consists of FMR1, FXR1, and FXR2. All three members bind RNA and contain sequence motifs that are commonly found in RNA-binding proteins, including two KH domains and an RGG box. The FMR1/FXR proteins also contain a 60S ribosomal subunit interaction domain and a protein-protein interaction domain which mediates homomer and heteromer formation with each family member. Nevertheless, the specific molecular functions of FMR1/FXR proteins are unknown. Here we report the cloning and characterization of a Drosophila melanogaster homolog of the mammalian FMR1/FXR gene family. This first invertebrate homolog, termed dfmr1, has a high degree of amino acid sequence identity/similarity with the defined functional domains of the FMR1/FXR proteins. The dfmr1 product binds RNA and is similar in subcellular localization and embryonic expression pattern to the mammalian FMR1/FXR proteins. Overexpression of dfmr1 driven by the UAS-GAL4 system leads to apoptotic cell loss in all adult Drosophila tissues examined. This phenotype is dependent on the activity of the KH domains. The ability to induce a dominant phenotype by overexpressing dfmr1 opens the possibility of using genetic approaches in Drosophila to identify the pathways in which the FMR1/FXR proteins function.     

Circadian rhythm-dependent alterations of gene expression in Drosophila brain lacking fragile X mental retardation protein

Fragile X syndrome is caused by the loss of the FMR1 gene product, fragile X mental retardation protein (FMRP). The loss of FMRP leads to altered circadian rhythm behaviors in both mouse and Drosophila; however, the molecular mechanism behind this phenomenon remains elusive. Here we performed a series of gene expression analyses, including of both mRNAs and microRNAs (miRNAs), and identified a number of mRNAs and miRNAs (miRNA-1 and miRNA-281) with circadian rhythm-dependent altered expression in dfmr1 mutant flies. Identification of these RNAs lays the foundation for future investigations of the molecular pathway(s) underlying the altered circadian rhythms associated with loss of dFmr1.     

Methylation of the arginine-glycine-rich region in the fragile X mental retardation protein FMRP differentially affects RNA binding

The C-terminal end of the fragile X mental retardation protein contains a stretch of amino acid residues that are enriched in arginine and glycine. Recent studies using recombinant FMRPs have demonstrated that this region participates in RNA binding in vitro, with calculated Kds ranging from 1-10 nM depending on the RNA. It is known that other arginine-glycine-rich proteins are subject to site-specific methylation by protein arginine methyltransferases (PRMTs) that are particularly abundant in most cells. We have demonstrated that the interaction of homoribopolymer mimetic RNAs with human FMRP (hFMRP) made in PRMT-containing cell-free lysates is more sensitive to increasing salt concentrations than recombinant hFMRP expressed in bacteria. We have also shown that blocking methylation with adenosine-2', 3'-dialdehyde (AdOx) alters homoribopolymer binding and hFMRP target mRNA binding; both increases and decreases are observed as a function of methylation. These data suggest that changes in PRMT activity that occur during development, or arise via signal transduction may be a means of regulating the binding of hFMRP to mRNA in vivo.