First in vivo evidence of microRNA-induced fragile X mental retardation syndrome.

Fragile X syndrome (FraX) is the most common form of inherited mental retardation, with the estimated prevalence of 30% of total human mental retardation disorders, and is also among the most frequent single gene disorders.1 The gene affected by the syndrome in 99% patients, FMR1, is transcriptionally inactivated by the expansion and the methylation of trinucleotide (CGG) repeats, located in the 5′-untranslated region (5′-UTR) of the gene.2 FMR1 encodes an RNA-binding protein, FMRP, which is associated with polyribosome assembly in an RNA-dependent manner and capable of suppressing protein translation through an RNA interference (RNAi)-like pathway that is important for neuronal development and plasticity. However, no appropriate animal model is available for the study of FraX etiology because current Drosophila and mouse models are all based on the gene deletion of FMRP, completely irrelevant to the mechanism of RNAi. Many recent studies have indicated that human FraX results from microRNA (miRNA)-mediated methylation in the CpG region of FMR1 rCGG expansion, which is targeted by a small RNA derived from the 3′-UTR of the FMR1 expanded allele transcript.3–5 Such dicer-processed miRNA may trigger the formation of RNA-induced initiator of transcriptional gene silencing (RITS) on the homologous r(CGG) repeats and leads to heterochromatin repression of the FMR1 locus. Thus, the etiological mechanism of FraX is owing to the miRNA-mediated genomic suppression in FMR1, rather than gene deletion. To investigate the role of miRNA in this proposed disease model, we have designed and tested man-made miRNA transgenes directed against the fish fmr1 gene to generate loss-of-function transgenic zebrafish. Like human, zebrafish possesses three FMRP-related genes, fmr1, fxr1 and fxr2, which are orthologous to the human FMR1, FXR1 and FXR2 genes, respectively.6 The expression patterns of these FMRP-familial genes in zebrafish tissues are broadly consistent with those in mouse and human, suggesting that such a loss-of-fmr1-function zebrafish is an excellent model organism for studying the FraX etiology.6 We constructed the anti-fmr1 miRNA transgene based on a proof-of-principle design of the artificial SpRNAi-rGFP transgene as previously reported in the generation of gene-knockout zebrafish.7 The miRNA was expressed under the control of a GABA(A) receptor βZ2 gene promoter in zebrafish brain and was directed against the nucleotides (nts) 25–45 region of the zebrafish fmr1 5′-UTR methylation site (Accession number NM152963). This target region contains several 5′-UTR r(CGG) repeats, reminiscent of the native anti-FMR1 miRNA target site in human FraX.5 As shown in Figure 1, fluorescent three-dimensional micrograph showed abnormal neuron morphology and connectivity in the loss-of-fmr1-function transgenics, similar to those in human FraX. In fish lateral pallium, wild-type neurons presented normal dendritic outline and well connection to each other (yellow arrows), whereas the transgenics exhibited thin, strip-shape neurons, reminiscent of the abnormal dendritic spine neurons in the human FraX.8,9 Altered synaptic plasticity has been reported to be a major physiological damage in the FraX of human and mouse, particularly in the hippocampal stratum radiatum area.9,10 Synapse deformity frequently occurred in the loss-of-fmr1-function neurons (red arrows), indicating the functional role of FMR1 in activity-dependent synaptic neuron plasticity. Further, the group 1 metabotropic glutamate receptor-activated long-term depression (LTD) could be augmented in the absence of fmr1, suggesting that exaggerated LTD may be responsible for aspects of abnormal neuronal responses in FraX, such as autism. Figure 1 Morphological changes of lateral pallium neurons in the loss-of-fmr1-function zebrafish. Because the whole Tg(UAS:gfp) zebrafish tissues expresses green GFP and the anti-fmr1 miRNA transgene is marked with red GFP, we can easily observe the normal dendritic ... With the advance of such an miRNA-mediated loss-of-fmr1-function zebrafish model, we may now investigate the molecular pathological and neurobehavioral changes that are common in human patients but difficult to be evidenced in the FMR1-deleted mice. The zebrafish FraX model established here is consistent with the hypothetic mechanism of the human FraX, in which the anti-fmr1 miRNA prevents synaptic strengthening and blocks local protein synthesis-dependent synaptic connections, a cascade of events for which FMR1 has been strongly implicated. As a result, future therapy and research based on this novel FraX model will be a great challenge.