Deimination restores inner retinal visual function in murine demyelinating disease
Mabel Enriquez-Algeciras, … , Vittorio Porciatti, Sanjoy K. Bhattacharya
Mabel Enriquez-Algeciras, … , Vittorio Porciatti, Sanjoy K. Bhattacharya
Published January 2, 2013
Citation Information: J Clin Invest. 2013;123(2):646-656. https://doi.org/10.1172/JCI64811.
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Research Article Autoimmunity

Deimination restores inner retinal visual function in murine demyelinating disease

Abstract

Progressive loss of visual function frequently accompanies demyelinating diseases such as multiple sclerosis (MS) and is hypothesized to be the result of damage to the axons and soma of neurons. Here, we show that dendritic impairment is also involved in these diseases. Deimination, a posttranslational modification, was reduced in the retinal ganglion cell layer of MS patients and in a transgenic mouse model of MS (ND4 mice). Reduced deimination accompanied a decrease in inner retinal function in ND4 mice, indicating loss of vision. Local restoration of deimination dramatically improved retinal function and elongation of neurites in isolated neurons. Further, neurite length was decreased by downregulation of deimination or siRNA knockdown of the export-binding protein REF, a primary target for deimination in these cells. REF localized to dendrites and bound selective mRNAs and translation machinery to promote protein synthesis. Thus, protein deimination and dendritic outgrowth play key roles in visual function and may be a general feature of demyelinating diseases.

Authors

Mabel Enriquez-Algeciras, Di Ding, Fabrizio G. Mastronardi, Robert E. Marc, Vittorio Porciatti, Sanjoy K. Bhattacharya

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Figure 4

Identification of REFBP2 and RNA binding.

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Identification of REFBP2 and RNA binding. (A) Western blot analysis of I...
(A) Western blot analysis of IP products from RGC lysates using anti-citrulline antibody. Lane 1 (S; starting material): lane 2 (+; anti-citrulline-IP). Arrow indicates the REFBP2 identification region of the gel. (B) Protein extract from RGCs were gel separated; REFBP2 (∼23 KDa) bands were excised, reloaded onto a fresh SDS-PAGE as indicated (upper panel), and detected using anti-REFBP2 and anti-citrulline antibodies. (C) Mass spectrometry (LTQ OrbitrapXL) identified deiminated REFBP2 arginines (bold). The bold (*) and italicized (**) arginines were found deiminated or methylated in different REFBP2 molecules. RNA binding pocket is underlined. (D) SDS-PAGE analysis of E. coli lysate without (–) and with (+) IPTG induction and purified recombinant REFBP2 (P). Bottom panel shows Western blot analysis using an anti-His antibody. (E) RNA-binding assay analyzed on a PAGE gel in TAE buffer. Total RNA used as starting material; bound RNA species eluted from control (–d; nondeiminated) and deiminated (+d) REFBP2. Arrowhead indicates enriched RNA species. (F) Purified TUNP or REFBP2 (top) was UV cross-linked with the total RNA pool and subjected to PCR for amplification of SNAP-25 mRNA (bottom). (G) Relative SNAP-25 protein amount produced by in vitro translation; (–d) and (+d) indicate control and deiminated REFBP2. (H) RGC neurite length upon treatment with control or shRNA-REF. Length (μm) measured were averaged from 40 neurons. *P < 0.05 (I) Localization of deimination (Cit) and REFBP2 in RGC neurites; (J) merged picture of DAPI (blue), anti-citrulline (green) and anti-REFBP2 (red). Arrows indicate colocalization of deimination and REFBP2. Scale bars: 25 μm.