Abnormal dendritic spines in fragile X knockout mice: Maturation and pruning deficits

Fragile X syndrome arises from blocked expression of the fragile X mental retardation protein (FMRP). Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. Here we describe 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 were longer than those in wild-type mice and were 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 was greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning.

Synaptic regulation of protein synthesis and the fragile X protein

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use “synaptoneurosomes,” a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P

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. We present here the identification of CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) 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 heteromerization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has recently 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.

Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that 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. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1–2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

Hairpins are formed by the single DNA strands of the fragile X triplet repeats: structure and biological implications.

Inordinate expansion and hypermethylation of the fragile X DNA triplet repeat, (GGC)n.(GCC)n, are correlated with the ability of the individual G- and C-rich single strands to form hairpin structures. Two-dimensional NMR and gel electrophoresis studies show that both the G- and C-rich single strands form hairpins under physiological conditions. This propensity of hairpin formation is more pronounced for the C-rich strand than for the G-rich strand. This observation suggests that the C-rich strand is more likely to form hairpin or "slippage" structure and show asymmetric strand expansion during replication. NMR data also show that the hairpins formed by the C-rich strands fold in such a way that the cytosine at the CpG step of the stem is C.C paired. The presence of a C.C mismatch at the CpG site generates local flexibility, thereby providing analogs of the transition to the methyltransferase. In other words, the hairpins of the C-rich strand act as better substrates for the human methyltransferase than the Watson-Crick duplex or the G-rich strand. Therefore, hairpin formation could account for the specific methylation of the CpG island in the fragile X repeat that occurs during inactivation of the FMR1 gene during the onset of the disease.

Evolution of the Friedreich’s ataxia trinucleotide repeat expansion: Founder effect and premutations

Friedreich’s ataxia, the most frequent inherited ataxia, is caused, in the vast majority of cases, by large GAA repeat expansions in the first intron of the frataxin gene. The normal sequence corresponds to a moderately polymorphic trinucleotide repeat with bimodal size distribution. Small normal alleles have approximately eight to nine repeats whereas a more heterogeneous mode of large normal alleles ranges from 16 to 34 GAA. The latter class accounts for ≈17% of normal alleles. To identify the origin of the expansion mutation, we analyzed linkage disequilibrium between expansion mutations or normal alleles and a haplotype of five polymorphic markers within or close to the frataxin gene; 51% of the expansions were associated with a single haplotype, and the other expansions were associated with haplotypes that could be related to the major one by mutation at a polymorphic marker or by ancient recombination. Of interest, the major haplotype associated with expansion is also the major haplotype associated with the larger alleles in the normal size range and was almost never found associated with the smaller normal alleles. The results indicate that most if not all large normal alleles derive from a single founder chromosome and that they represent a reservoir for larger expansion events, possibly through “premutation” intermediates. Indeed, we found two such alleles (42 and 60 GAA) that underwent cataclysmic expansion to pathological range in a single generation. This stepwise evolution to large trinucleotide expansions already was suggested for myotonic dystrophy and fragile X syndrome and may relate to a common mutational mechanism, despite sequence motif differences.

Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension.

Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. The resulting "knockout" mice have confirmed some hypotheses, have upset others, but have rarely been uninformative. Models of several human genetic diseases have been produced by targeting--including Gaucher disease, cystic fibrosis, and the fragile X syndrome. These diseases are primarily determined by defects in single genes, and their modes of inheritance are well understood. When the disease under study has a complex etiology with multiple genetic and environmental components, the generation of animal models becomes more difficult but no less valuable. The problems associated with dissecting out the individual genetic factors also increases substantially and the distinction between causation and correlation is often difficult. To prove causation in a complex system requires rigorous adherence to the principle that the experiments must allow detection of the effects of changing only a single variable at one time. Gene targeting experiments, when properly designed, can test the effects of a precise genetic change completely free from the effects of differences in any other genes (linked or unlinked to the test gene). They therefore allow proofs of causation.

An n-allele model for progressive amplification in the FMR1 locus.

An n-allele model is developed for the FMR1 locus, which causes the fragile X syndrome, where n is the number of triplet repeats in the first exon. Frequencies in the general population and in index families are used to generate an n to n + delta transition matrix that predicts specific risks in satisfactory agreement with observation. However, until sequencing distinguishes between stable and unstable alleles with the same value of n, it is premature to infer whether allelic frequencies at the FMR1 locus are at equilibrium or, as some have suggested, are evolving toward higher frequencies of the pathogenic allele.

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.

Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome

X-linked lymphoproliferative syndrome (XLP) is an inherited immunodeficiency characterized by increased susceptibility to Epstein–Barr virus (EBV). In affected males, primary EBV infection leads to the uncontrolled proliferation of virus-containing B cells and reactive cytotoxic T cells, often culminating in the development of high-grade lymphoma. The XLP gene has been mapped to chromosome band Xq25 through linkage analysis and the discovery of patients harboring large constitutional genomic deletions. We describe here the presence of small deletions and intragenic mutations that specifically disrupt a gene named DSHP in 6 of 10 unrelated patients with XLP. This gene encodes a predicted protein of 128 amino acids composing a single SH2 domain with extensive homology to the SH2 domain of SHIP, an inositol polyphosphate 5-phosphatase that functions as a negative regulator of lymphocyte activation. DSHP is expressed in transformed T cell lines and is induced following in vitro activation of peripheral blood T lymphocytes. Expression of DSHP is restricted in vivo to lymphoid tissues, and RNA in situ hybridization demonstrates DSHP expression in activated T and B cell regions of reactive lymph nodes and in both T and B cell neoplasms. These observations confirm the identity of DSHP as the gene responsible for XLP, and suggest a role in the regulation of lymphocyte activation and proliferation. Induction of DSHP may sustain the immune response by interfering with SHIP-mediated inhibition of lymphocyte activation, while its inactivation in XLP patients results in a selective immunodeficiency to EBV.

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

Increased levels of plasma lysosomal enzymes in patients with Lowe syndrome

Lowe syndrome is an X-linked disorder that has a complex phenotype that includes progressive renal failure and blindness. The disease is caused by mutations in an inositol polyphosphate 5-phosphatase designated OCRL. It has been shown that the OCRL protein is found on the surface of lysosomes and that a renal tubular cell line deficient in OCRL accumulated substrate phosphatidylinositol 4,5-bisphosphate. Because this lipid is required for vesicle trafficking from lysosomes, we postulate that there is a defect in lysosomal enzyme trafficking in patients with Lowe syndrome that leads to increased extracellular lysosomal enzymes and might lead to tissue damage and contribute to the pathogenesis of the disease. We have measured seven lysosomal enzymes in the plasma of 15 patients with Lowe syndrome and 15 age-matched male controls. We find a 1.6- to 2.0-fold increase in all of the enzymes measured. When the data was analyzed by quintiles of activity for all of the enzymes, we found that 95% of values in the lowest quintile come from normal subjects whereas in the highest quintile 85% of the values are from patients with Lowe syndrome. The increased enzyme levels are not attributable to renal insufficiency because there was no difference in enzyme activity in the four patients with the highest creatinine levels compared with the six patients with the lowest creatinine values.

WIP, a protein associated with Wiskott–Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells

Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.

Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.