Loss of AKAP1 triggers Drp1 dephosphorylation-mediated mitochondrial fragmentation and loss in retinal ganglion cells

Impairment of mitochondrial structure and function is strongly linked to glaucoma pathogenesis. Despite the widely appreciated disease relevance of mitochondrial dysfunction and loss, the molecular mechanisms underlying mitochondrial fragmentation and metabolic stress in glaucoma are poorly understood. We demonstrate here that glaucomatous retinal ganglion cells (RGCs) show loss of A-kinase anchoring protein 1 (AKAP1), activation of calcineurin (CaN) and reduction of dynamin-related protein 1 (Drp1) phosphorylation at serine 637 (S637). These findings suggest that AKAP1-mediated phosphorylation of Drp1 at S637 has a critical role in RGC survival in glaucomatous neurodegeneration. Male mice lacking AKAP1 show increases of CaN and total Drp1 level, as well as a decrease of Drp1 phosphorylation at S637 in the retina. Ultrastructural analysis of mitochondria shows that loss of AKAP1 triggers mitochondrial fragmentation and loss, as well as mitophagosome formation in RGCs. Loss of AKAP1 deregulates oxidative phosphorylation complexes (Cxs) by increasing CxII and decreasing CxIII-V, leading to metabolic and oxidative stress. Also, loss of AKAP1 decreases Akt phosphorylation at S473 and activates the Bim/Bax signaling pathway in the retina. These results suggest that loss of AKAP1 has a critical role in RGC dysfunction by decreasing Drp1 dephosphorylation at S637, deregulating OXPHOS, decreasing Akt phosphorylation and activating the Bim/Bax pathway in glaucomatous neurodegeneration. Thus, we propose that overexpression of AKAP1 or modulation of Drp1 phosphorylation at S637 are potential therapeutic strategies for neuroprotective intervention in glaucoma and other mitochondria-related optic neuropathies.


Introduction
Primary open-angle glaucoma (POAG) is characterized by a slow and progressive degeneration of retinal ganglion cells (RGCs) and their axons, leading to loss of visual function 1 . The factors contributing to degeneration of the RGC and its axon degeneration in POAG are not well understood. Recent studies have shown that POAG patients have mitochondrial abnormalities [2][3][4][5][6][7] .
Evidence from our group and others indicates that compromised mitochondrial dynamics, metabolic stress and mitochondrial dysfunction by glaucomatous insults such as elevated intraocular pressure (IOP) and oxidative stress are critical to RGC loss in experimental glaucoma [8][9][10][11][12][13][14][15][16][17][18][19] . Despite the widely appreciated disease relevance of mitochondrial dysfunction and loss, the molecular mechanisms underlying the impairment of mitochondrial structure and function in glaucoma are poorly understood.
Previous studies showed that inhibition of Drp1 activity prevents mitochondrial fission and protects RGCs and their axons in experimental glaucoma 14,43,44 . However, it remains unknown whether AKAP1-mediated Drp1 phosphorylation at S637 play a critical role in glaucomatous neurodegeneration. Along this line, the existence of AKAP1 in RGCs and its role in glaucoma are completely unknown.
To address these questions, we investigated AKAP1 protein expression and Drp1 phosphorylation at S637 in retinas from glaucomatous DBA/2J mice, and also evaluated the effect of AKAP1 loss in the retina using AKAP1 knockout (AKAP1 -/-) mice.

AKAP1 deficiency in glaucomatous RGCs.
To determine whether AKAP1 is altered in glaucomatous RGCs that were induced by IOP elevation, we examined AKAP1 protein expression in the retina of a mouse model of glaucoma DBA/2J mice, which spontaneously develop elevated IOP and glaucomatous damage with age 8,14,45 using Western blot and immunohistochemical analyses. Control DBA/2J-Gpnmb + /SjJ (D2-Gpnmb + ) was used in which the gene for Gpnmb that has been altered to wild type sequence by targeted gene mutation. It is otherwise identical to the DBA/2J mouse strain, but maintains normal IOP during aging and does not develop optic nerve axon loss 46,47 . Our results demonstrated for the first time that elevated IOP significantly decreased AKAP1 protein expression in the retinas of 10-month-old glaucomatous DBA/2J mice compared with agematched control D2-Gpnmb + mice (Fig. 1a). We observed that AKAP1 immunoreactivity was highly present in the outer plexiform layer (OPL) and ganglion cell layer (GCL) in D2-Gpnmb + mice (Fig. 1b). More specifically, AKAP1 immunoreactivity was colocalized with neuronal class III β-tubulin (TUJ1)-positive RGCs in the GCL of D2-Gpnmb + mice. Of interest, however, AKAP1 immunoreactivity was decreased in the OPL and TUJ1-positive RGCs in the GCL of glaucomatous DBA/2J mice compared with D2-Gpnmb + mice (Fig. 1b).
More importantly, AKAP1 protects brain neuronal cells against cerebral ischemic stroke by inhibiting Drp1-dependent mitochondrial fission 30 . Since elevated IOP increased CaN and total Drp1 protein expression 14,52 , as well as Drp1 inhibition rescued RGCs and their axons by preserving mitochondrial integrity in the retina and/or glial lamina of glaucomatous DBA/2J mice 14 , we examined the expression levels of CaN and total Drp1, as well as phosphorylation of Drp1 at S637 in the retina of of 10-month-old glaucomatous DBA/2J mice using Western blot and immunohistochemical analyses. We observed a significant increase of CaN protein level in the retina of glaucomatous DBA/2J mice compared with age-matched control D2-Gpnmb + mice ( Fig. 2a). Consistently, immunohistochemical analysis showed an increase of CaN immunoreactivity in RNA-binding protein with multiple splicing (RBPMS)-positive RGCs as well as neurons in the inner nuclear layer (INL) of glaucomatous DBA/2J mice compared with D2-Gpnmb + mice (Fig. 2b). We observed a significant increase of total Drp1 protein levels in the retina of glaucomatous DBA/2J mice compared with D2-Gpnmb + mice (Fig. 2c). Following the normalization of phospho-Drp1-S637 by total Drp1, however, we oberved a significant decrease of Drp1 phosphorylation at S637 in the retina of glaucomatous DBA/2J mice (Fig. 2c).
These results suggest that elevated IOP triggers CaN-dependent dephosphorylation of Drp1 at S637 in glaucomatous RGCs, leading to mitochondrial fragmentation 14 .

Loss of AKAP1 triggers CaN-mediated dephosphorylation of Drp1 at S637 in the retina
To determine whether loss of AKAP1 directly alters CaN and total Drp1 protein expression, as well as Drp1 phosphorylation in the retina, we used the retinas from mice lacking AKAP1 (AKAP1 -/-) 30 and examined the expression levels of CaN and total Drp1, as well as phosphorylation of Drp1-S637 and S616 using Western blot and immunohistochemical analyses.
Quantitative real-time RT-PCR analysis showed that AKAP1 gene expression was significantly decreased in the retina of AKAP1 -/mice (Fig. 3a). We observed a significant increase of CaN protein expression in the retina of AKAP1 -/mice compared with wild-type (WT) mice (Fig. 3b).
Consistent with this finding, increased CaN immunoreactivity was present in RBPMS-positive RGCs as well as neurons in the INL in AKAP1 -/mice compared with WT mice (Fig. 3c).
Interestingly, we observed a significant increase of total Drp1 protein expression in the retina of AKAP1 -/mice compared with WT mice (Fig. 4a). Following the normalization of phospho-Drp1-S637 and S616 by total Drp1, we also observed that AKAP1 loss induced a significant decrease of Drp1-S637 in the retina of AKAP1 -/mice, whereas there was no significant difference in Drp1-S616 phosphorylation in the retina between WT and AKAP1 -/mice (Fig. 4b).
Consistent with this finding, increased Drp1 immunoreactivity was present in RBPMS-positive RGCs in AKAP1 -/mice compared with WT mice (Fig. 4b).

AKAP1 loss causes mitochondrial fission and mitophagosome formation in RGCs
To determine whether loss of AKAP1 alters mitochondrial fusion activity in the retina and also triggers mitochondrial fission and loss in RGCs in AKAP1 -/mice, we examined the expression levels of optic atropy type 1 (OPA1) and mitofusin (Mfn) 1 and 2 in the retina as well as the alteration of mitochondrial ultrastructure using Western blot and electron microscope (EM) tomography analyses. We observed a significant decrease of total OPA1 protein expression in the retina of AKAP1 -/mice compared with WT mice (Fig. 5a). More specifically, small isoform (80 kDa) of OPA1 protein expression was significantly decreased, whereas there was no difference in the large isoform (100 kDa) of OPA1 protein expression in the retina of AKAP1 -/mice (Fig. 5a). We also observed a significant increase of Mfn1 protein expression, whereas there was no difference in Mfn2 protein expression in the retina of AKAP1 -/mice ( Fig. 5a) Using EM and EM tomography analyses, we further assessed changes of mitochondrial ultrastructure by measuring mitochondrial volume density, number, cross-sectional area and cristae abundance in RGC somas in AKAP1 -/mice. In comparison with WT mice, representative volumes generated by EM tomography provided evidence for mitochondrial fission and loss of mitochondrial mass (Fig. 5, b-d). Quantitative analysis showed significant decreases of mitochondrial volume density (13 ± 0.7%), and mitochondrial cross-sectional area (0.14 ± 0.02 µm 2 ), but a significant increase of mitochondrial number (0.92 ± 0.06 per µm 2 ) in RGC somas of AKAP1 -/mice compared with WT (22 ± 2 %, mitochondrial volume density; 0.20 ± 0.01 µm 2 , mitochondrial cross-sectional area ; 0.67 ± 0.05 per µm 2 , mitochondrial number) (Fig. 5c). However, there was no significant difference in mitochondrial cristae abundance, which is the ratio of cristae membrane surface area divided by the mitochondrial outer membrane surface area, in RGC somas between WT and AKAP1 -/mice (Fig. 5c). Using 3D reconstruction of tomographic volume, we observed that the mitochondria from AKAP1 -/mice are significantly smaller than the WT mitochondria. Smaller mitochondria should have fewer cristae, given that cristae shape doesn't change a lot, but the density of cristae was about the same between WT and AKAP1 -/- (Fig. 5d). These results suggest that AKAP1 loss induces an imbalance of mitochondrial dynamics by triggering mitochondrial fission and loss in RGCs.
AKAP1 loss promotes mitochondrial abnormalities and mitophagy in cardiac injury 53 . Based on our current findings of mitochondrial fission and loss, we determined whether AKAP1 loss induces mitophagosome formation using Western blot, immunohistochemistry and EM tomography analyses. Indeed, AKAP1 loss significantly enhanced LC3-II protein expression, but decreased p62 protein expression in the retina of AKAP1 -/mice compared with WT mice (Fig. 6a). Immunohistochemical analysis showed strong LC3 immunoreactivities in RGC somas and axons in the GCL of AKAP1 -/mice (Fig. 6b). Notably, segmented volumes from threedimensional (3D) tomographic reconstructions using EM tomography revealed examples of degraded mitochondria with severe cristae depletion that were engulfed in mitophagosomes in RGC soma of AKAP1 -/mice ( Fig. 6c-i).

AKAP1 loss results in OXPHOS dysfunction and induces oxidative stress in the retina
Since AKAP1 deletion increased superoxide production in primary hippocampal and cortical neurons in response to glutamate excitotoxicity as well as resulted in OXPHOS complex (Cx) II dysfunction 30 , we determined whether loss of AKAP1 alters OXPHOS Cxs and induces oxidative stress in the retina. Using OXPHOS rodent cocktail antibody, we further observed that AKAP1 loss significantly increases OXPHOS Cx II, but decreased OXPHOS Cx III-V protein expression in the retina compared with WT mice (Fig. 7a). However, there was no significant difference in OXPHOS Cx I protein expression in the retina between WT and AKAP1 -/mice ( Fig. 7a). We observed a significant increase of superoxide dismutase 2 (SOD2) protein expression in AKAP1 -/mice compared with WT mice (Fig. 7b). Immunohistochemical analysis using SOD2 antibody demonstrated that SOD2 protein expression was increased in the inner retina, including RGCs in the GCL, of AKAP1 -/mice compared with WT mice (Fig. 7c). These results suggest that AKAP1 loss compromises OXPHOS and may contribute to oxidative stress in the retina.

AKAP1 loss inactivates Akt and activates Bim/Bax pathway in the retina
To determine whether loss of AKAP1 alters Akt phosphorylation at serine 473 (S473), we examined the expression levels of Akt protein expression and Akt-S473 phosphorylation in AKAP1 -/mice using Western blot and immunohistochemical analyses. We observed a significant increase of total Akt protein expression, but decrease of phospho-Akt-S473 protein expression in AKAP1 -/mice compared with WT mice (Fig. 8a). Following the normalization of phospho-Akt-S473 by total Akt, we observe that AKAP1 loss induced a significant decrease of Akt-S473 phosphorylation in the retina of AKAP1 -/mice compared with WT mice (Fig. 8a). In addition, we observed that Akt immunoreactivity was increased in the INL and GCL of the retina

Discussion
We here demonstrated for the first time that RGCs showed AKAP1 immunoreactivity in D2-Gpnmb+ mice and elevated IOP triggered loss of AKAP1 in glaucomatous RGCs. In addition, elevated IOP increased CaN and total Drp1 levels in glaucomatous RGCs. It also impaired Drp1 phosphorylation at S637 in glaucomatous retina. These results suggest that AKAP1 and Drp1 phosphorylation at S637 have critical roles in RGC mitochondria and survival against glaucomatous damage. We also observed that loss of AKAP1 increased CaN and total Drp1 levels in RGCs, and compromised Drp1 phosphorylation at S637 in the retina, leading to mitochondrial fission and loss, and mitophagosome formation in RGCs. In addition, loss of AKAP1 resulted in OXPHOS dysfunction, as well as Akt inactivation and Bim/Bax activation; these effects contribute to glaucomatous neurodegeneration. Impairment of mitochondrial dynamics is critically involved in glaucomatous RGCs and their axon degeneration 8,11,14,43 , and inhibition of Drp1 rescues RGCs and their axons by preserving mitochondrial integrity in experimental glaucoma 14,43 . Our previous study demonstrated that elevated IOP showed a significant increase of total Drp1 level, while it did not alter phosphorylation of Drp1 at S616 in glaucomatous retina 14 ; these effects suggest that elevated IOP-mediated mitochondrial fission activity is not dependent on Drp1 phosphorylation at S616 in glaucomatous RGC degeneration. Hence, our novel finding that glaucomatous RGCs lacking AKAP1 trigger CaN-mediated dephosphorylation of Drp1 at S637 suggest an important rationale to further investigate the impact of AKAP1 loss, mitochondrial fragmentation and its related signaling pathway in RGCs.
AKAP1 is an important regulator of mitochondrial function by PKA anchoring and Drp1 phosphorylation at S637, and overexpression of AKAP1 is neuroprotective in neuronal cells against the mitochondrial toxin rotenone 25,27,29,30,50 . Emerging evidence showed that mice lacking AKAP1 exhibit increased infarct following transient cerebral ischemia as well as increased Drp1 localization to mitochondria from the forebrain tissues and decreased Drp1 phosphorylation at S637 30 . Our findings of enhanced CaN-mediated Drp1 phosphorylation at S637, and extensive mitochondrial fission and loss in the retina of AKAP1 -/mice suggest that AKAP1 loss directly contribute to Drp1 phosphorylation-dependent mitochondrial fragmentation and loss in glaucomatous RGCs. This notion is also supported by the evidence of increasing LC3-II and decreasing p62 levels, as well as mitophagosome formation that contains degrading mitochondria in RGC somas of AKAP1 -/mice. In addition, it is supported by the evidence of our previous report showing autophagosome/mitophagosome formation in RGC axons of glaucomatous DBA/2J mice 14 . Previous study indicated the notion that AKAP1 regulates mitochondrial dynamics by posttranslational modifications, but levels of Drp1, OPA1 and Mfn2 were unaltered in total forebrain homogenates from AKAP1 -/mice 30  Emerging evidence showed that AKAP1 deletion results in OXPHOS Cx II dysfunction in neuronal cells 30 . OXPHOS Cx II is a source of the mitochondrial reserve respiratory capacity that is regulated by metabolic sensor and promotes cell survival against hypoxia. In the present study, the observation that increasing OXPHOS Cx II, but decreasing OXPHOS Cxs III-V activities triggered by loss of AKAP1 in the retina is likely to imply that AKAP1 plays a critical role in OXPHOS function in RGCs. Moreover, increasing OXPHOS Cx II activity may be an important endogenous compensatory defense mechanism in response to oxidative stress.
Further, this imbalance of OXPHOS Cxs suggests another possibility that AKAP1 loss may contribute to an increase of ROS production and decrease of ATP production in the retina that are previously reported in pressure and/or oxidative stress-induced RGC degeneration 14 . In particular, association of POAG with polymorphism of mitochondrial cytochrome c oxidase subunit 1 of OXPHOS Cx IV suggests a potential role of abnormal OXPHOS-mediated mitochondrial dysfunction in glaucoma pathogenesis 3,4 . Therefore, these results collectively suggest that AKAP1 loss in glaucomatous retina contributes to OXPHOS dysfunction that is associated with metabolic and oxidative stress by increasing ROS production and decreasing ATP reduction, leading to RGC death during glaucomatous neurodegeneration.
Loss of AKAP1 inactivates Akt signaling and increases apoptosis in cardiac dysfunction 57, 58 . Akt pathway controls the expression of apoptosis-related gene through transcription factors such as FoxO (FoxO1 and FoxO3a) 59,60 and Akt activation promotes cell survival by inhibiting FoxO3a/Bim/Bax pathway 55,59,60 . The pro-apoptotic Bcl-2 homology domain 3-only protein Bim induces apoptosis, primarily through its increased binding activity toward multiple pro-survival Bcl-2-like proteins, whose dissociations activate Bak and Bax 61,62 .
In addition, Bax-mediated mitochondrial outer membrane permeabilization is an important pathophysiological mechanism for metabolic dysfunction and cell death 63,64 , and Bax activation plays a critical role in mitochondrial dysfunction-mediated RGC degeneration 14,17 . Because Drp1 contributes to Bax oligomerization, mitochondrial fission and the cellular apoptotic pathway 65-67 , increased total Drp1 and Bax levels in AKAP1 -/retinas suggest that AKAP1 loss may contribute to Drp1-mediated Bax oligomerization, leading to mitochondrial fission. Recent evidence from our group and others indicated that activation of cAMP/PKA pathway promotes RGC survival 55,68-70 and that AKAP1-mediated neuroprotection requires PKA anchoring and Drp1 phosphorylation at S637 27,29,30,50,71 . In this regard, our observation that AKAP1 -/retinas dephosphorylates Akt at S473 and activates Bim/Bax pathway raise the possibility that AKAP1 may be critical to RGC protection by modulating Akt/Bim/Bax pathway against glaucomatous damage. Future studies will be necessary to examine whether overexpression of AKAP1 rescues RGCs by promoting Drp1 phosphorylation at S637, maintaining metabolic activity, activating Akt pathway and inhibiting Bim/Bax pathway.
In summary, our results represent the first direct evidence of a Drp1 phosphorylationmediated mitochondrial pathogenic mechanism that leads to mitochondrial fragmentation and metabolic dysfunction in glaucomatous RGC degeneration. Also, we provide evidence for the first time that AKAP1 loss-mediated CaN activation and Drp1 dephosphorylation at S637 result in mitochondrial fragmentation, mitophagosome formation, OXPHOS dysfunction and oxidative stress, as well as Akt inactivation and Bim/Bax activation in RGCs. Therefore, we propose overexpression of AKAP1 and modulation of Drp1 phosphorylation at S637 as therapeutic strategies for neuroprotective intervention in glaucoma and other mitochondria-related optic neuropathies.

Animals
Adult female DBA/2J and D2-Gpnmb + mice (The Jackson Laboratory), and adult male WT and AKPA1 -/mice were housed in covered cages, fed with a standard rodent diet ad libitum, and kept on a12 h light/12 h dark cycle. The following PCR primers were used to determine AKAP1

RT-PCR and quantitative real-time RT-PCR
Total RNA was isolated from retinas using the Absolutely RNA Miniprep Kit (Stratagene), according to the manufacturer's protocol as previously described 55  Output data were obtained as Ct values and the differential mRNA expression of AKAP1 among groups was calculated using the comparative Ct method. GAPDH mRNA, an internal control, was amplified along with the target gene, and the Ct value of GAPDH was used to normalize the expression of the target gene.

Transmission electron microscopy
For conventional EM, two eyes from each group (n = 2 mice) were fixed via cardiac perfusion with 2% paraformaldehyde, 2.5% glutaraldehyde (Ted Pella, Redding, CA) in 0.15M sodium cacodylate (pH 7.4, Sigma) solution at 37°C and placed in pre-cooled fixative of the same composition on ice for 1 h. The following procedure was used to optimize mitochondrial structural preservation and membrane contrast 72

Electron microscope tomography
Sections of retina tissues from each group were cut at a thickness of 400 nm. For each reconstruction, a double-tilt series of images at one degree tilt increment was collected with an FEI titan hibase electron microscope operated at 300 kV. Images were recorded with a Gatan 4Kx4K CCD camera. The magnification was 11,000x and the pixel resolution was 0.81 nm.
The IMOD package was used for alignment, reconstruction and volume segmentation. Volume segmentation was performed by manual tracing of membranes in the planes of highest resolution with the Drawing Tools and Interpolator plug-ins 73 . The mitochondrial reconstructions and surface-rendered volumes were visualized using 3DMOD. Measurements of mitochondrial outer, inner boundary, and cristae membrane surface areas and volumes were made within segmented volumes using IMODinfo. These were used to determine the cristae abundance, defined as the ratio: sum of the cristae membrane surface areas divided by the mitochondrial outer membrane surface area. Movies of the tomographic volume were made using IMODmovie.

Statistical analyses
Data were shown as mean ± S.D. Comparison of experimental conditions was evaluated using the two-tailed unpaired Student's t-test between groups. P < 0.05 was considered to be statistically significant.  (c) Western blot analyses for total Drp1 and phospho-Drp1 S637 in the retinas of glaucomatous DBA/2J and age-matched D2-Gpnmb + mice. (d) Representative images from immunohistochemical analyses for total Drp1 (green, arrowheads) co-labeled with TUJ1 (red, arrowheads) in RGCs. Note that glaucomatous RGCs showed increases of total Drp1 protein expression. Blue color indicates nucleus. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Mean ± SD; n = 3 (a and c); **P < 0.01 and ***P < 0.001 (two-tailed Student's t-test). Scale bar: 20 µm.  Representative images from immunohistochemical analyses for total Drp1 (green, arrowheads) co-labeled with RBPMS (red, arrowheads) in RGCs. Note that glaucomatous RGCs showed an increase of total Drp1 protein expression in RGCs. Blue color indicates nucleus. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Mean ± SD; n = 3 or 6 (a); *P < 0.05 (two-tailed Student's t-test). Scale bar: 20 µm. (b) 1.6-nm thick slices through the middle of tomographic volumes of WT RGC somas show an abundance of larger mitochondria. In contrast, the mitochondria are smaller with a greater number in AKAP1 -/-RGC somas, but show no alteration of cristae architecture. (c) Quantitative analyses of mitochondrial volume density, number and cross-sectional area and cristae abundance in WT and AKAP1 -/-RGC somas. Note that AKAP1 -/-RGC somas show significant loss of mitochondrial mass and cross-sectional area, and a significant increase in mitochondrial number. However, there was no significant difference in cristae abundance of AKAP1 -/-RGC somas. (d) Top view of the surface-rendered volume showing 3 adjacent mitochondria from a WT tomographic volume (left) and an AKAP1 -/volume (right). Comparing the side view with the top view provides perspective on the distribution of the predominantly tubular cristae (in an assortment of colors) and reveals substantial heterogeneity of cristae size. The mitochondrial outer membranes were made translucent to better visualize the cristae. The number of cristae in each mitochondrion is indicated. Mean ± SD (a) and Mean ± SEM (c); n = 3 (a), n = 11 (mitochondrial volume, number and cristae abundance for WT and AKAP1 -/-, c) and n = 22 or 39 (mitochondrial size for WT or AKAP1 -/-, c); *P < 0.05 and **P < 0.01 (two-tailed Student's ttest). Scale bars: 200 nm. showing the crista and right-hand portion of the IBM inside the mitochondrion, which is inside the mitophagosome. (i) An oblique view with the mitophagosome and outer mitochondrial membranes made transparent to see the crista and two IBM fragments. Note that mitochondria in the AKAP1 -/-RGC soma demonstrate autophagosome/mitophagosome formation. Mean ± SD; n = 3 or 4 (a); *P < 0.05 and **P < 0.01 (two-tailed Student's t-test). Scale bar: 500 nm. (c) Representative images from immunohistochemical analyses for SOD2 (green, arrowheads) co-labeled with Brn3a (red, arrowheads) in the retina. Note that AKAP1 -/retina shows increases of SOD2 protein expression in the inner retinal layer compared with WT retina. Blue color indicates nucleus. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Mean ± SD; n = 4 or 10 (a) and n = 4 (b). *P < 0.05 and **P < 0.01 (two-tailed Student's t-test). Scale bar: 20 µm.