A Defective Crosstalk Between Neurons and Müller Glial Cells in the rd1 Retina Impairs the Regenerative Potential of Glial Stem Cells

Abstract

Müller glial cells (MGC) are stem cells in the retina. Although their regenerative capacity is very low in mammals, the use of MGC as stem cells to regenerate photoreceptors (PHRs) during retina degenerations, such as in retinitis pigmentosa, is being intensely studied. Changes affecting PHRs in diseased retinas have been thoroughly investigated; however, whether MGC are also affected is still unclear. We here investigated whether MGC in retinal degeneration 1 (rd1) mouse, an animal model of retinitis pigmentosa, have impaired stem cell properties or structure. rd1 MGC showed an altered morphology, both in culture and in the whole retina. Using mixed neuron-glial cultures obtained from newborn mice retinas, we determined that proliferation was significantly lower in rd1 than in wild type (wt) MGC. Levels of stem cell markers, such as Nestin and Sox2, were also markedly reduced in rd1 MGC compared to wt MGC in neuron-glial cultures and in retina cryosections, even before the onset of PHR degeneration. We then investigated whether neuron-glial crosstalk was involved in these changes. Noteworthy, Nestin expression was restored in rd1 MGC in co-culture with wt neurons. Conversely, Nestin expression decreased in wt MGC in co-culture with rd1 neurons, as occurred in rd1 MGC in rd1 neuron-glial mixed cultures. These results imply that MGC proliferation and stem cell markers are reduced in rd1 retinas and might be restored by their interaction with "healthy" PHRs, suggesting that alterations in rd1 PHRs lead to a disruption in neuron-glial crosstalk affecting the regenerative potential of MGC.

Keywords: Müller glial cells; photoreceptors; retinal degeneration; retinal regeneration; stem cells.

FIGURE 1

Identification of neurons and Müller glial cells (MGC) in glial, neuronal and mixed neuron-glial cultures. Phase (a,f,k) and fluorescence (b–e,g–j,l–o) photomicrographs of wt MGC culture (a–e), 2-day wt neuronal cultures (f–j) and 6-day wt mixed neuron-glial cultures (k–o), showing MGC (thick arrows) identified by their anti-Glutamine Synthase (GS) labeling (b), with their actin cytoskeleton visualized by phalloidin labeling (c,m); photoreceptors (thin arrows) labeled with anti-CRX (h,l) and amacrine neurons (arrowheads) labeled with HPC-1 (g) antibodies. Cell nuclei were visualized with the DNA probe DAPI (d,i,n). Note that cell nuclei were markedly smaller in neurons than in MGC (n). Merge images (e,j,o). Scale bars: 20 μm.

FIGURE 2

Selective degeneration of photoreceptors in the rd1 mice retina and in mixed rd1 neuron-glial cultures. Panel (A) phase (a,d,g,j,m,p) and fluorescence (b,c,e,f,h,i,k,l,n,o,q,r) photomicrographs of retinal cryosections from wt PND 9 (a–c), PND 14 (g–i) and Adult (m–o, PND30); and rd1 PND 9 (d–f), PND 14 (j–l), and Adult (p–r). Photoreceptors were labeled with an anti-opsin, Rho4-D2, antibody (c,f,i,l,o,r) and cell nuclei were stained with the nuclear probe TOPRO-3 (b,e,h,k,n,q). White vertical bars show the thickness of the Outer Nuclear Layer (ONL) in wt and rd1 retinas. Opsin immunolabeling was evident in wt and rd1 retinas at PND 9 (c,f), and concentrated in photoreceptor outer segments of wt retinas at PND 14 and 30 (i,o); in contrast, it was barely visible in rd1 retinas at PND 14 (l) and completely disappeared by PND 30 (r). White arrows in (m,o) show outer segments in wt photoreceptors at PND 30. Panel (B) phase (a,e) and fluorescence (b–d,f–h) photomicrographs of mixed neuron-glial cultures from wt (a–d) and rd1 (e–h) mice after 11 days in culture showing apoptotic neurons (white arrowheads) detected by TUNEL labeling (b,f). Nuclei were stained with DAPI (c,g). Note that MGC nuclei (empty arrowheads in a–h) remained intact in wt and rd1 cultures. Bars in (i) show the percentage of neurons with fragmented or pyknotic nuclei, (j) depict the percentage of TUNEL-positive neurons, (k) show the percentage of surviving MGC and (l) represent the fold-change in Bax mRNA levels relative to these levels in wt cultures at day 6. In Panel (A) scale bar: 50 μm. In Panel (B) scale bar: 5 μm. Results represent the mean ± SEM of three experiments (n = 3). Statistical analysis was performed using a two-tailed Student t-test [Panel (B) j,k] and a One-way ANOVA with a post hoc Tukey test [Panel (B) i,l]. ^*p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 3

Decreased proliferation of rd1 Müller glial cells in neuron-glial cultures. Phase (a,f) and fluorescence photomicrographs of 11-day wt (a–e) and rd1 (f–j) mixed neuron-glial cultures showing BrdU uptake by MGC, determined with a monoclonal anti-BrdU antibody (b,g, arrows). MGC were identified by their labeling with an anti-GS antibody (c,h). Nuclei were labeled with DAPI (d,i). Merge (e,j). Bars in (k) represent the percentage of BrdU-positive at days 6 and 11. Scale bar: 30 μm. Results represent the mean ± SEM of three experiments (n = 3). Statistical analysis was performed using a One-way ANOVA with a post hoc Tukey test. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 4

Nestin expression decreased in rd1 retinas. Panel (A) fluorescence photomicrographs of 11-day wt (a–d) and rd1 (e–h) mixed neuron-glial cultures showing NESTIN (a,e); GS (b,f) and TOPRO-3 (c,g) labeling. Merge pictures are shown in (d,h). Bars in (i) depict the percentage of NESTIN-expressing MGC, determined by flow cytometry. The histogram (j) shows the distribution of the intensity of NESTIN expression in MGC and bars in (k) show the medium fluorescence intensity of NESTIN labeling in wt and rd1 MGC. Bars represent the fold-change in Nestin (l) and Vimentin mRNA levels (m) in rd1 and wt cultures, relative to their levels in wt cultures at day 6. Scale bar: 40 μm. Results represent the mean ± SEM of three experiments (n = 3). Panel (B) Fluorescence photomicrographs of wt (a–d) and rd1 (e–h) PND 14 retina cryosections showing MGC labeling with NESTIN (a,e) and GS (b,f); MGC projections are indicated with thin arrows. Nuclei were stained with TOPRO-3 (c,g). Merge pictures are shown in (d,h). Confocal optical sections of retinas show the regular nestin filament distribution in MGC radial processes in wt retinas (i, arrowheads) and the conspicuous thickening and disorganization of radial processes at their basal ends in the rd1 retinas (j, arrowheads). Bars in (k) show the fold change in the number of NESTIN-expressing MGC per area. Bars (l) depict the Corrected Total Cell Fluorescence (CTCF) as Integrated Density− (Area of selected cells x Mean fluorescence of background readings, analyzed after manually outlining regions of interest (ROI) with the software Fiji-Image J (see section “Materials and Methods”). Bars in (m) depict the thickness of MGC radial processes in wt and rd1 retinas [shown with thin arrows in Panel (B) a,b,d,e,f,h; arrowheads in i,j]. Bars in (n) represent the fold changes in Nestin mRNA levels at PND 0-180 in rd1 and wt mice retinas relative to their levels at PND 0 in wt retinas. Scale bar: 20 μm. Results in k–m represents the mean ± SEM of ten separate measures. Results in (n) represent the mean ± SEM of three mice per condition at each PND day analyzed. Statistical analysis was performed using a two-tailed Student t-test [Panels (A) i,k and (B) k–m] and a One-way ANOVA with a post hoc Tukey test [Panels (A) l,m and (B) n]. ^*p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 5

SOX2 expression decreased in rd1 retinas. Fluorescence photomicrographs show SOX2 expression (a,d, arrowheads) in retina cryosections obtained from wt (a–c) and rd1 (d–f) mice at PND14, determined with an anti-SOX2 polyclonal antibody. Nuclei were stained with DAPI (b,e) and merge pictures are shown in (c,f). Bars (g) show the fold change of total SOX2-positive nuclei in rd1 retinas relative to wt retinas. Bars (h) represent the fold changes in Sox2 mRNA levels at PND 0-180 in rd1 and wt mice retinas relative to their levels at PND 0 in wt retinas. Scale bar: 20 μm. Results in (g) represent the mean ± SEM of ten measures. Results in (h) represent the mean ± SEM of three mice per condition. Statistical analysis was performed using a two-tailed Student t-test (g), and a One-way ANOVA with a post hoc Tukey test (h). ^*p < 0.05, ^∗∗∗p < 0.001.

FIGURE 6

Changes in Nestin expression in MGC when co-cultured with wt or rd1 neurons. Neuron-glial co-cultures were prepared seeding unlabeled rd1 MGC (Panel A) or wt MGC (Panel B) over 3-day cultures of either rd1 or wt neurons labeled with the green fluorescent CellTracer probe (thin arrows), and were then incubated for 24 h. Fluorescence photomicrographs show Cell-Trace labeled neurons (green in a,e) and NESTIN-labeled MGC (red in b,f, arrowheads); nuclei were stained with TOPRO-3 (blue in c,g). Merge pictures are shown in (d,h). Bars in Panels A (i) and B (i) show the fold change in NESTIN-positive rd1 MGC (Panel A) or wt MGC (Panel B) when co-cultured with either wt neurons or rd1 neurons, relative to pure rd1 or wt MGC cultures, respectively. Bars in Panels A (j) and B (j) depict the fold change in Nestin mRNA levels in rd1 MGC (Panel A) or wt MGC (Panel B) when co-cultured with either wt or rd1 neurons, relative to pure rd1 or wt MGC cultures, respectively. Scale bar: 40 μm. Results represent the mean ± SEM of three experiments (n = 3). UD, undetected. Statistical analysis was performed using a One-way ANOVA with a post hoc Tukey test. ^*p < 0.05, ^∗∗p < 0.01.

FIGURE 7

MGC morphology and neuron:glial ratio was altered in rd1 mixed neuron-glial cultures. Phase (a,e) and fluorescence photomicrographs (b–d,f–h) of 6-day wt (a–d) and rd1 (e–h) mixed neuron glial cultures showing neuronal cells (arrowheads) growing on top of MGC (arrows). Actin cytoskeleton was stained with phalloidin (b,f) and nuclei with DAPI (c,g). Merge pictures are shown in (d,h). Scale bar: 50 μm.