Daniela C. Zarnescu

Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway

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. Here we show that in vivo mammalian FMRP interacts 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, we show that AGO1 is critical for FMRP function in neural development and synaptogenesis. Our results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease.

Identification of small molecules rescuing fragile X syndrome phenotypes in Drosophila

Fragile X syndrome is caused by the functional loss of the fragile X mental retardation 1 (FMR1) gene. Deletion of the FMR1 ortholog in Drosophila melanogaster (Fmr1) recapitulates many phenotypes associated with fragile X syndrome. We have discovered that Fmr1 mutant Drosophila die during development when reared on food containing increased levels of glutamate, which is consistent with the theory that FMR1 loss results in excess glutamate signaling. Using this lethal phenotype, we screened a chemical library of 2,000 compounds and identified nine molecules that rescued the lethality, including three that implicate the GABAergic inhibitory pathway. Indeed, GABA treatment rescued several known Fmr1 mutant phenotypes in flies, including mushroom bodies defects, excess Futsch translation and abnormal male courtship behavior. These data are consistent with GABAergic inhibition of the enhanced excitatory pathway in fragile X syndrome. In addition, our screen reveals that the muscarinic cholinergic receptors may have a role in fragile X syndrome in parallel to the GABAergic pathway. These results point to potential therapeutic approaches for treating fragile X syndrome.

Crumbs interacts with moesin and βHeavy-spectrin in the apical membrane skeleton of Drosophila

The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, we report that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst mutants that eliminate the apical SBMS. Our data indicate that it is specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs that is essential to efficiently recruit both the apical SBMS and the FERM domain protein, DMoesin. Crumbs, Discs lost, βHeavy-spectrin, and DMoesin are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. We propose that Crumbs stabilizes the apical SBMS via DMoesin and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity.

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS

The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders.

Fragile X protein controls the efficacy of mRNA transport in Drosophila neurons

Fragile X syndrome, the most common form of inherited mental retardation is caused by mutations in the FMR1 gene. FMR1 encodes an RNA-binding protein thought to control the transport and translation of target mRNAs. While the function of FMRP in translational control has been clearly demonstrated, its role in mRNA transport and localization in neurons remains elusive. Using a genetically encoded mRNA imaging system in Drosophila we provide the first demonstration that FMRP controls mRNA transport. Live imaging of FMRP associated mRNAs show that mRNA granules are less motile and exhibit decreased directional movement in dFmr1 mutant neurons. Furthermore, Fluorescence Recovery After Photobleaching experiments show that the mobile fraction of mRNA molecules within neurites is dependent on FMRP dosage. These data support a model whereby FMRP regulates transport efficacy, by regulating the association between mRNA cargo and microtubules and suggest a new mechanism for the disease.

Futsch/MAP1B mRNA Is a Translational Target of TDP-43 and Is Neuroprotective in a Drosophila Model of Amyotrophic Lateral Sclerosis

TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. Here we provide evidence that TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where we find higher levels of transcript compared with controls. TDP-43 also leads to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulates the translation of futsch mRNA. We also show that futsch overexpression is neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal cords in a manner similar to our observations in Drosophila motor neurons. Together, our results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo.

Fragile X Protein Controls Neural Stem Cell Proliferation in the Drosophila Brain

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X protein (FMRP), an RNA-binding protein thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs. We have recently shown that FMRP is required to control the proliferation of the germline in Drosophila. To determine whether FMRP is also required for proliferation during brain development, we examined the distribution of cell cycle markers in dFmr1 brains compared with wild-type throughout larval development. Our results indicate that the loss of dFmr1 leads to a significant increase in the number of mitotic neuroblasts (NB) and BrdU incorporation in the brain, consistent with the notion that FMRP controls proliferation during neurogenesis. Developmental studies suggest that FMRP also inhibits neuroblast exit from quiescence in early larval brains, as indicated by misexpression of Cyclin E. Live imaging experiments indicate that by the third instar larval stage, the length of the cell cycle is unaffected, although more cells are found in S and G2/M in dFmr1 brains compared with wild-type. To determine the role of FMRP in neuroblast division and differentiation, we used Mosaic Analysis with a Repressible Marker (MARCM) approaches in the developing larval brain and found that single dFmr1 NB generate significantly more neurons than controls. Our results demonstrate that FMRP is required during brain development to control the exit from quiescence and proliferative capacity of NB as well as neuron production, which may provide insights into the autistic component of FXS.

Come FLY with us: toward understanding fragile X syndrome

The past few years have seen an increased number of articles using Drosophila as a model system to study fragile X syndrome. Phenotypic analyses have demonstrated an array of neuronal and behavioral defects similar to the phenotypes reported in mouse models as well as human patients. The availability of both cellular and molecular tools along with the power of genetics makes the tiny fruit fly a premiere model in elucidating the molecular basis of fragile X syndrome. Here, we summarize the advances made in recent years in the characterization of fragile X Drosophila models and the identification of new molecular partners in neural development.

Drosophila Fragile X Protein controls cellular proliferation by regulating cbl levels in the ovary

FMRP is an RNA binding protein linked to the most common form of inherited mental retardation, Fragile X syndrome (FraX). In addition to severe cognitive deficits, FraX etiology includes postpubescent macroorchidism, which is thought to result from overproliferation. Using a Drosophila FraX model, we show that FMRP controls germline proliferation during oogenesis. dFmr1 null ovaries contain egg chambers with both fewer and supranumerary germ cells. The mutant germaria contain a significantly increased number of cyclin E and PhosphoHistone H3 positive cells, suggesting that loss of FMRP leads to defects in cell cycle progression. BrdU incorporation and flow cytometry data suggest that, in addition to proliferation, germline endoreplication and ploidy are also affected by the loss of FMRP during ovary development. Here we report that FMRP controls the levels of cbl mRNA in the ovary and that reducing cbl gene dosage by half rescues the dFmr1 oogenesis phenotypes. These data support a model whereby FMRP controls germline proliferation by regulating the expression of cbl in the developing ovary.

Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis.

SUMMARY Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by complex neuronal and glial phenotypes. Recently, RNA-based mechanisms have been linked to ALS via RNA-binding proteins such as TDP-43, which has been studied in vivo using models ranging from yeast to rodents. We have developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of pathology, including motor neuron loss, locomotor dysfunction and reduced survival. Here we report the phenotypic consequences of expressing wild-type and four different ALS-linked TDP-43 mutations in neurons and glia. We show that TDP-43-driven neurodegeneration phenotypes are dose- and age-dependent. In motor neurons, TDP-43 appears restricted to nuclei, which are significantly misshapen due to mutant but not wild-type protein expression. In glia and in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta. TDP-43-containing RNA granules are motile in cultured motor neurons, although wild-type and mutant variants exhibit different kinetic properties. At the neuromuscular junction, the expression of TDP-43 in motor neurons versus glia leads to seemingly opposite synaptic phenotypes that, surprisingly, translate into comparable locomotor defects. Finally, we explore sleep as a behavioral readout of TDP-43 expression and find evidence of sleep fragmentation consistent with hyperexcitability, a suggested mechanism in ALS. These findings support the notion that although motor neurons and glia are both involved in ALS pathology, at the cellular level they can exhibit different responses to TDP-43. In addition, our data suggest that individual TDP-43 alleles utilize distinct molecular mechanisms, which will be important for developing therapeutic strategies.