Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina - PubMed (original) (raw)
Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina
Yoshiki Kuse et al. Sci Rep. 2016.
Abstract
Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU(+) cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin(+) photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway.
Figures
Figure 1. ASC-CM promoted retinal photoreceptor differentiation following MNU-induced retinal damage.
(A) Experimental procedure. Mice were injected MNU and BrdU by intraperitoneal (i.p.) injection. Following this the mice were treated with 150-fold concentrated vehicle (DMEM) or ASC-CM by intravitreal (i.v.) injection. At 2 and 4 days post MNU treatment, mice were similarly treated with vehicle or ASC-CM. After 5 days, mice eyes were enucleated. (B–D) Typical immunostaining images showing rhodopsin (green) and BrdU (red) staining. BrdU+ cell were not observed in the ONL in the normal group. Some BrdU+ cells were observed in the control group and ASC-CM increased the number of BrdU+ cells in the ONL. ASC-CM also increased the number of rhodopsin+ BrdU+ cells. An enlarged image shows the presence of rhodopsin+ BrdU+ cells in the ONL. Data are shown as means ± S.E.M. (n = 6). ##p < 0.01 and #p < 0.05 vs. control (Student’s _t_-test). Scale bar = 20 μm.
Figure 2. PGRN increased BrdU+ cell numbers following light-induced retinal damage.
(A) Experimental procedure. Mice were exposed to visible light (8,000 lux) for 3 h. After light exposure, mice were administered BrdU by intraperitoneal (i.p.) injection and mice were treated with vehicle (PBS) or PGRN 2 μL by intravitreal (i.v.) injection. At 2 and 4 days post light-induced retinal damage, mice were similarly treated with vehicle or PGRN. After 5 days, mice eyes were enucleated. (B,D) Typical images of immunostaining showing rhodopsin (green) and BrdU (red). BrdU+ cells were not observed in the normal group. Some BrdU+ cells were observed in the control group and PGRN increased the number of BrdU+ cells specifically in the ONL. Close-up images indicate BrdU+ cells were rhodopsin negative. (C) The localization of Rx mRNA by in situ hybridization (ISH). Rx mRNA was colocalized with Rx protein and BrdU in PGRN-treated ONL but not control group as shown in close-up view. Data are shown as means ± S.E.M. (n = 9). ##p < 0.01 and #p < 0.05 vs. control (Student’s _t_-test). Scale bar = 20 μm, 10 μm.
Figure 3. The effect of PGRN on retinal precursor cells in primary culture.
(A) The eyes from 8-day old mice were enucleated and the retinas were dissected. After dissection the retinas were centrifuged with any reagents. The retinal cells were incubated for 20 h after dissociation. After incubation, the medium was changed and vehicle or PGRN (500 ng/mL) was added to the retinal cell culture. After 3 days, reagents were added to the culture. The cells were collected for western blotting (after 4 days) and for immunostaining (after 5 days). (B) The presence of precursor cells in the primary retinal cell culture was confirmed by immunostaining for DCX (neural precursor cells), CRX (photoreceptor precursor cells) and nestin (neural precursor cells). The images show DCX (green), CRX (red), nestin (magenta) and Hoechst 33342 (cyan) staining. (C–F) PGRN decreased the number of DCX+ cells and CRX+ cells compared to controls. Data are shown as means ± S.E.M. (n = 4). #p < 0.05 vs. control (Student’s _t_-test). (G,H) Typical immunostaining images showing rhodopsin (red) and Hoechst 33342 (cyan). PGRN treatment increased the number of rhodopsin+ cells compared to control. Data are shown as means ± S.E.M. (n = 3 or 4). ##p < 0.01 vs. control (Student’s _t_-test). (I) PGRN increased the number of rhodopsin+ photoreceptor cells and resulted in a decrease in the number of CRX+ photoreceptor precursor cells and DCX+ neural precursor cells. C: Control; P: PGRN.
Figure 4. SU11274 treatment inhibited the promotion of differentiation of photoreceptor cells by PGRN.
(A) After dissociation and incubation, the medium was changed and SU11274 (an HGFR inhibitor) 1 μM and vehicle and PGRN (500 ng/mL) were added in retinal cell culture. After 3 days, the medium was changed maintaining the same composition of additives. The cells were collected for western blotting and for immunostaining after 5 days. (B) Immunoblots showing that PGRN increased the phosphorylation level of HGFR following a 5 min incubation. SU11274 prevented the PGRN-induced increase in phosphorylation. (C,D) Typical immunostaining images showing rhodopsin (red) and Hoechst 33342 (cyan) staining. PGRN increased the number of rhodopsin+ cells compared to control. This effect was blocked by the addition of SU11274. Data are shown as means ± S.E.M. (n = 6 or 7). #p < 0.05 vs. control, *p < 0.05 vs. PGRN (Student’s _t_-test). (E,F) Typical blots and quantitative data demonstrating the increase of rhodopsin expression associated with PGRN treatment and the suppression of the increase by SU11274. Treatment with SU11274 alone had no effect on rhodopsin expression level. Data are shown as means ± S.E.M. (n = 3 or 4). #p < 0.05 vs. control, *p < 0.05 vs. PGRN (Student’s _t_-test). C: Control; P: PGRN; SU: SU11274.
Figure 5. PGRN loss causes the developmental disorder in the ONL.
(A,B) No expression of progranulin in the retina was observed in PGRN knockout (_Grn_−/−) mice. (C) ONL thickness was decreased in _Grn_−/− mice compared to wild-type mice. Data are shown as means ± S.E.M. (n = 4 to 6). ##p < 0.01 and #p < 0.05 vs. WT (Student’s _t_-test). (D) Rhodopsin expression was also decreased in Grn−/− mice. Data are shown as means ± S.E.M. (n = 5). #p < 0.05 vs. WT (Student’s _t_-test). WT: Wild-type; Hz: Heterozygous; KO: Knockout. Scale bar = 50 μm.
Figure 6. A putative association between PGRN and retinal regeneration.
Retinal injury induces the dedifferentiation of Müller glia. Müller glial reprogramming generates retinal precursor cells. Generated retinal precursor cells have the potential to migrate to any retinal layer and differentiate into various retinal cells. PGRN can promote the migration of retinal precursor cells to the ONL and encourage their differentiation to photoreceptor cells.
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