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. 2015 Dec 21;589(24 Pt B):3959-68.
doi: 10.1016/j.febslet.2015.11.037. Epub 2015 Nov 26.

Uncoupling Phototoxicity-Elicited Neural Dysmorphology and Death by Insidious Function and Selective Impairment of Ran-binding Protein 2 (Ranbp2)

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Free PMC article

Uncoupling Phototoxicity-Elicited Neural Dysmorphology and Death by Insidious Function and Selective Impairment of Ran-binding Protein 2 (Ranbp2)

Kyoung-in Cho et al. FEBS Lett. .
Free PMC article

Abstract

Morphological disintegration of neurons is coupled invariably to neural death. In particular, disruption of outer segments of photoreceptor neurons triggers photoreceptor death regardless of the pathological stressors. We show that Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) mice with mutations in SUMO-binding motif (SBM) of cyclophilin-like domain (CLD) of Ran-binding protein 2 (Ranbp2) expressed in a null Ranbp2 background lack untoward effects in photoreceptors in the absence of light-stress. However, compared to wild type photoreceptors, light-stress elicits profound disintegration of outer segments of Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) with paradoxical age-dependent resistance of photoreceptors to death and genotype-independent activation of caspases. Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) exhibit photoreceptor death-independent changes in ubiquitin-proteasome system (UPS), but death-dependent increase of ubiquitin carrier protein 9(ubc9) levels. Hence, insidious functional impairment of SBM of Ranbp2's CLD promotes neuroprotection and uncoupling of photoreceptor degeneration and death against phototoxicity.

Keywords: Cyclophilin-like domain; Neuroprotection; Photoreceptor neuron; Phototoxicity; Ran-binding protein 2; SUMOylation.

Figures

Fig. 1
Fig. 1
(A) Structural domains of Ranbp2. V/I-X-V/I-V/I is the consensus SUMO-binding motif (SBM) in the overlapping region (O) of CLD and IR1 of Ranbp2. Numbering refers to residues of the primary mouse sequence of Ranbp2 and positioning of selective domains and motifs of Ranbp2. Domains are not drawn to scale. (B) Transgenic BAC construct (~160 kb) of Ranbp2, Tg-Ranbp2CLDm-HA, with the mutations, I2471K andV2472A, in the SBM of CLD, and a C-terminal hemagglutinin (HA) tag insertion at the end of the terminal exon encoding the CY domain of Ranbp2. Note drawing not to scale. Legend: LD, leucine-rich domain; RBDn=1–4, Ran GTPase-binding domains, n=1–4; ZnFn =7, zinc finger-rich domains; KBD, kinesin-1-binding domain; CLD, cyclophilin-like domain; IR1 and IR2, internal repeats 1 and 2, respectively; M, middle domain between IR1 and IR2; O, overlapping region between CLD and IR1; CY, cyclophilin domain; UTR, untranslated region.
Fig. 2
Fig. 2
Morphological changes of photoreceptor neurons between wild-type and Ranbp2−/−::Tg-Ranbp2CLDm-HA mice reared in the absence and presence of light-stress. There are no visible morphological changes under light (A, C) and ultrastructural microscopy (B, D) of photoreceptors of wild-type (A, B) and Ranbp2−/−::Tg-Ranbp2CLDm-HA mice (C, D) reared under non-light treatment (NLT) at 24 weeks of age (A–D). Under light-treatment (E–J), the OS of photoreceptors of 24-week-old wild-type mice show mild disruption primarily at the base of the OS as shown by light (E) and ultrastructure microscopy (F, G), whereas light (H) and ultrastructure microscopy (I, J) show profuse derangement and erosion of the OS of photoreceptors, respectively, of age-matched Ranbp2−/−::Tg-Ranbp2CLDm-HA mice. Inset picture is magnified dashed box in (G). Legend: OS, outer segments of photoreceptors; IS, inner segments of photoreceptors; ONL, outer nuclear (cell bodies) layer of photoreceptors; OPL, outer plexiform layer; INL, inner nuclear (cell bodies) layer of retinal neurons; IPL, inner plexiform layer; GC, ganglionic neurons; RPE, retinal pigment epithelium; NLT, non-light treatment; LT, light treatment (light-stress); +/+, wild type; −/−::Tg-Ranbp2CLDm-HA, Ranbp2−/−::Tg-Ranbp2CLDm-HA. Scale bars: 20 μm (A, C, E, H), 5 μm (B, D, F, I), 1 μm (G, J).
Fig. 3
Fig. 3
Ranbp2−/−::Tg-Ranbp2CLDm-HA suppresses the development of apoptotic cell bodies by light-stress. The number of apoptotic cell bodies in 24-week-old Ranbp2−/−::Tg-Ranbp2CLDm-HA and wild type mice are scarce in the absence of light-stress and there is no difference between genotypes (A, B). In the presence of light stress (C, D), 24-week-old Ranbp2−/−::Tg-Ranbp2CLDm-HA show strong suppression of TUNEL+-apoptotic cell bodies (arrowheads) of photoreceptors in the central and peripheral regions of the retina compared to age-matched wild type mice. (B) and (D) are quantitative analyses of representative images in (A) and (C), respectively. Retinal sections were counterstained with DAPI. Data shown represent the mean ± S.D., n=5–6. Legend: NLT, non-light treatment; LT, light treatment (light stress); ONL, outer nuclear (cell bodies) layer of photoreceptors; +/+, wild type; −/-::Tg-Ranbp2CLDm-HA, Ranbp2−/−::Tg-Ranbp2CLDm-HA; n.s., non-significant; Scale bar: 20 μm.
Fig. 4
Fig. 4
Apoptotic cell bodies of photoreceptors of 24-week-old Ranbp2−/−::Tg-Ranbp2CLDm-HA and wild type mice lack differences in caspases’ activations (cleaved caspases) induced by light-stress. There are no changes in TUNEL+Casp3+, TUNEL+Casp7+, TUNEL+Casp8+, TUNEL+Casp9+ in cell bodies of photoreceptors between age-matched genotypes by light stress (A, B). Arrows show TUNEL+ cell bodies without caspases’ activations, whereas arrowheads show TUNEL+Casp+ cell bodies. (B) are quantitative analyses of representative images in (A). Retinal sections were counterstained with DAPI. Data shown represent the mean ± S.D., n=3. Legend: +/+, wild type; −/−::Tg-Ranbp2CLDm-HA, Ranbp2−/−::Tg-Ranbp2CLDm-HA; n.s., non-significant; Scale bar: 20 μm.
Fig. 5
Fig. 5
Age-dependent accumulation of ubiquitylated substrates and diubiquitin, and 20S proteasome activities, between Ranbp2−/−::Tg-Ranbp2CLDm-HA and wild type mice exposed to light-stress. In comparison to wild type mice, Ranbp2−/−::Tg-Ranbp2CLDm-HA mice present decreased and increased levels of ubiquitylated substrates and diubiquitin, respectively, at 8 weeks of age (A, C), but their levels are similar between genotypes at 24 weeks of age (B, C). (D) Retinal extracts of 24 week-old Ranbp2−/−::Tg-Ranbp2CLDm-HA and wild type mice have similar chymotrypsin and trypsin-like activities of the 20S proteasome, but Ranbp2−/−::Tg-Ranbp2CLDm-HA have modestly lower caspase-like activity than wild type mice. Data shown represent the mean ± S.D., n=4–6. Legend: +/+, wild type; −/−::Tg-Ranbp2CLDm-HA, Ranbp2−/−::Tg-Ranbp2CLDm-HA; di-Ub, diubiquitin; poly-UbP, poly-ubiquitylated proteins; hsc70, heat shock cytosolic protein 70; n.s., non-significant.
Fig. 6
Fig. 6
Age-dependent changes in S1 subunit and ubc9 proteostasis between Ranbp2−/−::Tg-Ranbp2CLDm-HA and wild type mice exposed to light-stress. (A) Diagram of the S1 and ubc9 partners of CLD and IR1 of Ranbp2, respectively. In comparison to wild type mice, Ranbp2−/−::Tg-Ranbp2CLDm-HA mice have increased levels of S1 subunit of the 19S cap of the 26S proteasome (B) and ubc9 (C) selectively at 8 and 24 weeks of age, respectively, whereas the levels of HDAC4 remain unchanged between genotypes at 8 and 24 weeks of age (D). Data shown represent the mean ± S.D., n=4. Legend: +/+, wild type; −/−::Tg-Ranbp2CLDm-HA, Ranbp2−/−::Tg-Ranbp2CLDm-HA; hsc70, heat shock cytosolic protein 70; n.s., non-significant.

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