Site-directed RNA repair of endogenous Mecp2 RNA in neurons.

Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from MECP2 gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G > A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of Mecp2 mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.

C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation.

The repressor element 1 (RE1) silencing transcription factor (REST) in stem cells represses hundreds of genes essential to neuronal function. During neurogenesis, REST is degraded in neural progenitors to promote subsequent elaboration of a mature neuronal phenotype. Prior studies indicate that part of the degradation mechanism involves phosphorylation of two sites in the C terminus of REST that require activity of beta-transducin repeat containing E3 ubiquitin protein ligase, ßTrCP. We identify a proline-directed phosphorylation motif, at serines 861/864 upstream of these sites, which is a substrate for the peptidylprolyl cis/trans isomerase, Pin1, as well as the ERK1/2 kinases. Mutation at S861/864 stabilizes REST, as does inhibition of Pin1 activity. Interestingly, we find that C-terminal domain small phosphatase 1 (CTDSP1), which is recruited by REST to neuronal genes, is present in REST immunocomplexes, dephosphorylates S861/864, and stabilizes REST. Expression of a REST peptide containing S861/864 in neural progenitors inhibits terminal neuronal differentiation. Together with previous work indicating that both REST and CTDSP1 are expressed to high levels in stem cells and down-regulated during neurogenesis, our results suggest that CTDSP1 activity stabilizes REST in stem cells and that ERK-dependent phosphorylation combined with Pin1 activity promotes REST degradation in neural progenitors.

Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction.

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.

Fine tuning of growth factor signals depends on fibrillin microfibril networks.

Growth factors, potent regulators of cell differentiation, tissue morphogenesis, tissue homeostasis, and cellular response to injury, reside in the extracellular matrix. Genetic evidence in humans and mice as well as biochemical data implicate fibrillins and LTBPs in the extracellular control of TGFbeta and BMP signaling. Fibrillins and LTBPs form tissue-specific and temporally regulated microfibril networks. In the developing embryo, three fibrillins and four LTBPs contribute molecular heterogeneity to microfibril networks, and provide different templates upon which TGFbeta-related growth factors can be positioned. By accommodating this molecular heterogeneity, microfibril architecture can orchestrate a variety of different signals in very specific tissue locations. Human fibrillinopathies display a broad phenotypic spectrum from tall to short stature, from hypermobile joints to joint contractures and stiffness, and from severe to mild or no cardiovascular manifestations. A spectrum of growth factor dysregulation may be caused by differential effects of mutations in fibrillins on microfibril architecture, thus altering appropriate targeting or positioning of growth factors within microfibril networks. Growth factor dysregulation may help to explain the broad phenotypic spectrum of the fibrillinopathies.

Differential expression of fibrillin-3 adds to microfibril variety in human and avian, but not rodent, connective tissues.

The human genome contains three fibrillins: FBN1 and FBN2, both well characterized, and FBN3, reported only as a cDNA sequence. Like FBN2, the highest expression levels of FBN3 were found in fetal tissues, with only low levels in postnatal tissues. Immunolocalization demonstrated fibrillin-3 in extracellular microfibrils abundant in developing skeletal elements, skin, lung, kidney, and skeletal muscle. Unlike the other two fibrillins, FBN3 expression is high in brain, and FBN3 is alternatively spliced, removing the exon encoding cbEGF2. Like FBN1, FBN3 contains three alternate exons in the 5' UTR. While FBN3 orthologs were identified in cow and chicken, Fbn3 appears to have been inactivated in the mouse genome, perhaps during chromosome fission events. Located on chromosome 19p13.3-13.2, FBN3 is a candidate gene for Weill-Marchesani syndrome.

Fibrillins can co-assemble in fibrils, but fibrillin fibril composition displays cell-specific differences.

Fibrillins are microfibril-forming extracellular matrix macromolecules that modulate skeletal development. In humans, mutations in fibrillins result in long bone overgrowth as well as other distinct phenotypes. Whether fibrillins form independent microfibrillar networks or can co-polymerize, forming a single microfibril, is not known. However, this knowledge is required to determine whether phenotypes arise because of loss of singular or composite functions of fibrillins. Immunolocalization experiments using tissues and de novo matrices elaborated by cultured cells demonstrated that both fibrillins can be present in the same individual microfibril in certain tissues and that both fibrillins can co-polymerize in fibroblast cultures. These studies suggest that the molecular information directing fibrillin fibril formation may be similar in both fibrillins. Furthermore, these studies provide a molecular basis for compensation of one fibrillin by the other during fetal life. In postnatal tissues, fibrillin-2 antibodies demonstrated exuberant staining in only one location: peripheral nerves. This surprising finding implicates distinct functions for fibrillin-2 in peripheral nerves, because a unique feature in humans and in mice mutant for fibrillin-2 is joint contractures that resolve over time.