Loss of Shp2-mediated mitogen-activated protein kinase signaling in Muller glial cells results in retinal degeneration

Abstract

Extensive studies have identified many growth factors and intracellular pathways that can promote neuronal survival after retinal injury, but the intrinsic survival mechanisms in the normal retina are poorly understood. Here we report that genetic ablation of Shp2 (Ptpn11) protein phosphatase resulted in progressive apoptosis of all retinal cell types. Loss of Shp2 specifically disrupted extracellular signal-regulated kinase (ERK) signaling in Müller cells, leading to Stat3 activation in photoreceptors. However, neither inactivation of Stat3 nor stimulation of AKT signaling could ameliorate the Shp2 retinal degeneration. Instead, constitutively activated Kras signaling not only rescued the retinal cell numbers in the Shp2 mutant but also functionally improved the electroretinogram recording (ERG). These results suggest that Shp2-mediated Ras-mitogen-activated protein kinase (Ras-MAPK) signaling plays a critical role in Müller cell maturation and function, which is necessary for the survival of retinal neurons.

Fig. 1.

Severe degeneration of the retina and optic nerve in Six3-Cre; Shp2^flox/flox adult mice. (A and B) Normal retinal histology in the Six3-Cre; Shp2^flox/flox mutant at postnatal day 7 (P7). (C to F) In the Six3-Cre; Shp2^flox/flox adult mice, the loss of optic nerve was observed in the dissected eyeballs (arrow in panel D) and in the transverse sections (asterisks indicate the vacuoles in the mutant optic nerve in panel F). (G to J) The Six3-Cre; Shp2^flox/flox mutant retina sections were approximately 50% thinner than that of the wild type at P21 (H), and the outer nuclear layer (ONL) was completely absent in some retinal regions at P120 (the arrow in panel J indicates the neighboring residual photoreceptor cells). Notice the photoreceptor cell rosette (arrowheads in panel H) and the lack of the optic nerve fiber layer/inner limiting membrane layer (NFL/ILML) in the mutant retina (arrow in panel H). GCL, ganglion cell layer. Bars, 40 μm.

Fig. 2.

Shp2 depletion in Six3-Cre; Shp2^flox/flox adult retina induced reduction in all retinal cell types and Müller cell gliosis. (A to F) Expression and relative fold changes of the retinal cell markers at P21. Brn3a stained for retinal ganglion cells, Pax6 for amacrine cells, Chx10 for bipolar cells, calbindin for horizontal cells, glutamine synthetase for Müller glial cells, recoverin for photoreceptors (rod and cone). The relative fold changes were determined by counting the number of cells and comparing them to the value for the wild type. All retina cell types were decreased by about 50% compared to wild type (bar graphs in panels A to F) (**, P ≤ 0.001). (G to I) We also observed the lack of optic nerve fiber by NF-165 staining (arrows in panels G), thinner inner plexiform layer (IPL) indicated by VC1.1 staining (for membranes of amacrine cells; brackets in panels H), and Müller cell gliosis by glial fibrillary acidic protein (GFAP) staining in the inner retina (arrows in panels I). (J and K) Expression of cell type markers in P120 Six3-Cre; Shp2^flox/flox mutants. Note the fewer and disorganized Müller glial cells in the mutant (J) and a complete cell loss in some ONL regions (brackets in panels K). DAPI, 4′,6′-diamidino-2-phenylindole. Bar, 50 μm.

Fig. 3.

Aberrant downstream signaling in the Six3-Cre; Shp2^flox/flox retina. (A) Western blotting confirmed Shp2 depletion in the Six3-Cre; Shp2^flox/flox retina at P5, but only by P21 did we observe the decrease in extracellular signal-regulated kinase (ERK) phosphorylation and the increase in Stat3 phosphorylation. The phospho-AKT level was unchanged. WT, wild type; pERK, phosphorylated ERK. (B) In the wild-type retina, Shp2 was ubiquitously expressed from the ganglion cell layer (GCL) to the inner nuclear layer (INL) but was almost absent in the outer nuclear layer (ONL). In the Six3-Cre; Shp2^flox/flox retina, Shp2 was depleted in all layers. (C) Phospho-Stat3 was undetectable in the wild-type retina but strongly elevated in the Six3-Cre; Shp2^flox/flox retina. Notice the scattered pattern of phospho-Stat3 staining (arrow). (D) The intensity of phospho-AKT staining was unchanged in the Six3-Cre; Shp2^flox/flox retina. (E to H) In the wild-type retina, phospho-ERK (pERK) staining was predominantly observed in glutamine synthetase (GS)-positive Müller glial cells (arrows). The white boxes in panels E show the sections shown in panels F, G, and H. Such staining was lost in the Six3-Cre; Shp2^flox/flox retina (arrowheads). Bar, 100 μm.

Fig. 4.

Stat3 ablation aggravated Shp2 retinal defects. (A) Stat3 depletion in the Six3-Cre; Stat3^flox/flox mutant retina did not affect ERK and AKT phosphorylation. In the Six3-Cre; Shp2^flox/flox; Stat3^flox/flox compound mutants, both Stat3 and ERK phosphorylation were disrupted. (B to E) Elimination of Stat3 did not cause an obvious retinal phenotype in the P21 Six3-Cre; Stat3^flox/flox mutant, but it accelerated retinal degeneration in the Six3-Cre; Shp2^flox/flox; Stat3^flox/flox compound mutants compared to the Six3-Cre; Shp2^flox/flox mutants. The sections were stained with hematoxylin and eosin (H&E). (F to I) Phospho-Stat3 was induced in the Six3-Cre; Shp2^flox/flox mutant and abolished in the Six3-Cre; Shp2^flox/flox; Stat3^flox/flox mutant. The arrowheads in panel G indicate phospho-Stat3-positive cells. Bar, 40 μm.

Fig. 5.

Pten mutation did not rescue Shp2 retinal defects. (A) Mutation in the gene encoding Pten, an upstream repressor of AKT signaling, resulted in ectopic AKT activation in the Six3-Cre; Shp2^flox/flox; Pten^flox/flox mutant, but phospho-ERK levels remained suppressed. (B to E) The optic nerve became enlarged in the Six3-Cre; Pten^flox/flox mutant but remained thin in the Six3-Cre; Shp2^flox/flox; Pten^flox/flox mutant. (F to I) At P21, Pten depletion led to thicker inner plexiform layers with displaced nuclei (brackets in panels H and I), but it failed to rescue retinal degeneration in the Six3-Cre; Shp2^flox/flox; Pten^flox/flox mutant. (J to M) Phospho-AKT remained elevated in the Six3-Cre; Shp2^flox/flox; Pten^flox/flox mutant. Bar, 40 μm.

Fig. 6.

Kras activation restored ERK phosphorylation and retinal morphology in the Six3-Cre; LSL-Kras^G12D; Shp2^flox/flox compound mutants. (A) As shown by Western blotting, expression of the activated Kras^G12D allele in the Six3-Cre; LSL-Kras^G12D; Shp2^flox/flox mutant restored the phospho-ERK level to that of the wild type and suppressed phospho-Stat3 expression, while phospho-AKT levels remained unchanged. (B to G) The optic nerve morphology and histology was rescued in the Six3-Cre; LSL-Kras^G12D; Shp2^flox/flox mutant. (H to M) While Shp2 staining remained absent in the Six3-Cre; LSL-Kras^G12D; Shp2^flox/flox mutant, phospho-ERK expression was restored in the Müller glial cells (arrows). (N to S) The retinal histology in the P21 Six3-Cre; LSL-Kras^G12D; Shp2^flox/flox mutant was comparable to that of the wild type. Notice the recovery of the optic nerve fiber layer/inner limiting membrane layer (arrows). (T) By cell counts, densities of all cell types recovered to the wild-type levels in the Six3-Cre; LSL-Kras^G12D; Shp2^flox/flox compound mutants. Bar, 50 μm.

Fig. 7.

Functional recovery of the electrophysiological responses in the Six3-Cre; LSL-Kras^G12D; Shp2^flox/flox retina. (A to C) Scotopic (rod-dominated) electroretinogram recording (ERG) responses to single flashes of light. (A) Representative traces at four increasing flash energies; (B) representative saturated A-wave traces; (C), rod B-wave amplitude versus flash energy with Naka-Rushton fitted curves. (D and E) Double-flash ERGs to isolate cone responses. (D) Representative traces in response to second flash; (E) average cone B-wave amplitudes for each of the three cohorts. Three-month-old adult mice were used for the ERG tests. The means ± standard errors of the means (SEM) (error bars) are shown.