Overexpression of Down syndrome cell adhesion molecule impairs precise synaptic targeting

Journal name:
Nature Neuroscience
Volume:
16,
Pages:
677–682
Year published:
DOI:
doi:10.1038/nn.3396
Received
Accepted
Published online

Abstract

Fragile X syndrome is caused by the loss of Fragile X mental retardation protein (FMRP), an RNA-binding protein that suppresses protein translation. We found that FMRP binds to Down syndrome cell adhesion molecule (Dscam) RNA, a molecule that is involved in neural development and has been implicated in Down syndrome. Elevated Dscam protein levels in FMRP null Drosophila and in flies with three copies of the Dscam gene both produced specific and similar synaptic targeting errors in a hard-wired neural circuit, which impaired the flies' sensory perception. Reducing Dscam levels in FMRP null flies reduced synaptic targeting errors and rescued behavioral responses. Our results indicate that excess Dscam protein may be a common molecular mechanism underlying altered neural wiring in intellectual disabilities such as Fragile X and Down syndromes.

At a glance

Figures

  1. FMRP suppresses Dscam protein translation.
    Figure 1: FMRP suppresses Dscam protein translation.

    (a) FMRP binds Dscam mRNA. FMRP-mRNA complexes were immunoprecipitated from Drosophila larval brains and specific targets were identified by reverse transcription (RT) PCR. FMRP has been previously shown to bind both its own mRNA and Futsch. No mRNAs were immunoprecipitated from Fmr1null mutants (Fmr1null IP), and Dscam mRNA did not immunoprecipitate with ELAV, another neuronal RNA-binding protein. (b) Loss of FMRP in Fragile X mutants increased neuronal Dscam protein levels. Representative fluorescent immunoblots of Dscam, FMRP and actin in different genotypes are shown. Protein samples for Dscamnull flies were prepared from embryos and showed restricted expression of FMRP isoforms. Dscam and FMRP protein intensities were normalized to actin (plotted in arbitrary units, a.u.), and the averages from nine experiments are shown. Errors bars represent s.e.m. (c) Quantitative real-time PCR analysis revealed that Dscam mRNA levels were not significantly altered in Fmr1null mutants, P > 0.05. Dscam mRNA for all experimental genotypes was measured as fold change from wild-type levels. The averages from six experiments are shown. Error bars are s.d. of the mean.

  2. The pSc mechanosensory neuron is identifiable between flies on the basis of the location of its corresponding bristle.
    Figure 2: The pSc mechanosensory neuron is identifiable between flies on the basis of the location of its corresponding bristle.

    (ad) The pSc neuron expressed both FMRP and Dscam. A cross section through a pSc bristle is shown in brightfield (a), and the corresponding FMRP immunofluorescence (green) within the pSc neuron (arrow) is shown in b. No detectable FMRP signal was observed in Fmr1null flies (c). Colocalization of FMRP and Dscam mRNA was observed in pSc neurons using fluorescence in situ hybridization for Dscam mRNA (magenta) combined with fluorescence immunohistochemistry for FMRP (green) (d). Arrowheads point to Dscam mRNA puncta, arrow points to FMRP signal. Nuclei are stained in blue. Scale bars represent 20 μm. (e) A single mechanosensory neuron innervates a single bristle. The axonal projection into the CNS of the right pSc mechanosensory neuron is shown in red. (f,g) The stereotyped synaptic connectivity of the pSc neuron was used as a readout for synaptic targeting errors. The wild-type pSc axonal arbor had a complex and stereotyped branching pattern (f). Quantitative analysis of wild-type pSc axons revealed 16 core branches (yellow lines) and 2 variable branches occurring in 50% of flies (blue lines). Individual branches of the pSc axonal arbor could be identified between flies, and their lengths and variance were quantified (g). Black lines represent the average lengths of each branch, red lines represent the s.d., and values are expressed in μm.

  3. Elevated Dscam protein levels produce specific axonal targeting errors.
    Figure 3: Elevated Dscam protein levels produce specific axonal targeting errors.

    (a) Ectopic branch number and length were increased in Fmr1null and Dscam X3 flies. (b) The core pSc skeleton did not change in branch number or lengths among different genotypes. (c,d) Axonal branch targeting was impaired in Fragile X mutant flies. Compared with the stereotyped axonal branching pattern of wild-type pSc neurons (c), flies lacking FMRP (d) had specific targeting errors, such as misrouting and aberrant midline crossing branches (arrows). The dotted lines mark the midline of the CNS. Scale bar represents 50 μm. (e) Dscam X3 flies had targeting errors (arrows) similar to those observed in Fragile X mutant flies. (f) Reducing Dscam levels in Fragile X mutants decreased targeting errors. Double mutant flies had a single null allele of Dscam and were homozygous null for Fmr1. (g) The frequency and type of targeting errors phenocopied between Fmr1null and Dscam X3 flies could be rescued by reducing Dscam protein levels. Frequency of occurrence for ten error types that were significantly greater than in wild type for both Fragile X mutants and Dscam X3 flies is shown. Double mutant flies had a significant reduction in five axonal targeting errors (purple rectangles). Statistical significance comparisons to wild type are indicated directly above the experimental genotypes' bar; the double mutant comparison to Fmr1null flies are indicated above a connecting line. All error bars represent s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, and NS indicates not significant (P > 0.05).

  4. Errors in synaptic targeting impair touch perception.
    Figure 4: Errors in synaptic targeting impair touch perception.

    (a) Mechanical stimulation of the pSc bristles using a controlled amount of fluorescent dye elicited a cleaning reflex from the rear legs. Transfer of the fluorescent dye from the back of the fly onto the rear legs was used to confirm a positive response. (b) Synaptic targeting of a single, identified neuron could be matched to the specific behavioral output for each fly. Representative images of the axonal arbors of previously stimulated pSc neurons are shown. Axonal arbors of mutant flies that either succeeded or failed to respond to bristle stimulation were compared with control responding flies. Arrows indicate targeting errors. The dotted line marks the midline. Scale bar represents 50 μm. (c) Synaptic targeting errors in the pSc neuron impaired touch perception in the Fragile X mutant and Dscam X3 flies and could be restored in the double mutant. The frequency of response is shown for mosaic flies with FMRP knocked down only in the scutellar neurons and for flies with three copies of Dscam, compared with their specific genetic controls (see Online Methods). The frequency of response to touch in mosaic double mutants, Dscamnull/455-Gal4; UAS-dsRNA-Fmr1, was significantly higher than mosaic Fragile X mutants (P < 0.05), and was not significantly different from controls (P > 0.05, n > 120 flies for each genotype). *P < 0.05, **P < 0.01. Error bars are s.e.m.

  5. FMRP binds multiple Dscam isoforms.
    Figure 5: FMRP binds multiple Dscam isoforms.

    (a) Three large arrays of alternatively spliced exons in Drosophila Dscam (exon 4, red; exon 6, blue; exon 9, green) encode for different extracellular immunoglobulin domains (Ig2, Ig3 and Ig7). Mutually exclusive splicing from each variable exon can produce 19,008 different extracellular domains. Exon 17 encodes for two alternate transmembrane domains (TM) and exons 19 and 23 can be included or excluded in the intracellular domain. (b) High-throughput pyrosequencing of Dscam bound to FMRP identified all possible Dscam isoforms. Dscam isoform distributions from a representative sequencing experiment of >1.2 million reads are shown as heatmaps for variable exons 4, 6 and 9. Isoform distributions from the input and the FMRP immunoprecipitation from three separate experiments are shown. Dscam RNA isoforms immunoprecipitated with FMRP showed no substantial bias in representation compared with Dscam isoforms in the input fraction, indicating that FMRP bound to all neuronal isoforms equally well.

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Author information

  1. These authors contributed equally to this work.

    • Vedrana Cvetkovska &
    • Alexa D Hibbert

Affiliations

  1. Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, Québec, Canada.

    • Vedrana Cvetkovska,
    • Alexa D Hibbert,
    • Farida Emran &
    • Brian E Chen
  2. Departments of Medicine, and Neurology and Neurosurgery, McGill University, Montréal, Québec, Canada.

    • Brian E Chen

Contributions

B.E.C. designed the experiments and supervised the project. V.C., A.D.H., F.E. and B.E.C. performed the experiments and analyzed the data. V.C., F.E. and B.E.C. wrote the manuscript.

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The authors declare no competing financial interests.

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