Loss of AKAP1 triggers Drp1 dephosphorylation-mediated mitochondrial fission and loss in retinal ganglion cells - PubMed (original) (raw)

doi: 10.1038/s41419-020-2456-6.

Guy A Perkins # 2, Keun-Young Kim # 2, YeEun Kong 1, Yonghoon Lee 1, Soo-Ho Choi 3, Yujia Liu 4, Dorota Skowronska-Krawczyk 1, Robert N Weinreb 1, Linda Zangwill 1, Stefan Strack 4, Won-Kyu Ju 5

Affiliations

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

Genea Edwards et al. Cell Death Dis. 2020.

Abstract

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 (Ser637). These findings suggest that AKAP1-mediated phosphorylation of Drp1 at Ser637 has a critical role in RGC survival in glaucomatous neurodegeneration. Male mice lacking AKAP1 show increases in CaN and total Drp1 levels, as well as a decrease in Drp1 phosphorylation at Ser637 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 (OXPHOS) complexes (Cxs) by increasing CxII and decreasing CxIII-V, leading to metabolic and oxidative stress. Also, loss of AKAP1 decreases Akt phosphorylation at Serine 473 (Ser473) and threonine 308 (Thr308) 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 phosphorylation at Ser637, deregulating OXPHOS, decreasing Akt phosphorylation at Ser473 and Thr308, and activating the Bim/Bax pathway in glaucomatous neurodegeneration. Thus, we propose that overexpression of AKAP1 or modulation of Drp1 phosphorylation at Ser637 are potential therapeutic strategies for neuroprotective intervention in glaucoma and other mitochondria-related optic neuropathies.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1

Fig. 1. AKAP1 deficiency in glaucomatous RGCs.

a Western blot analysis for AKAP1 in the retinas of 10-month-old glaucomatous DBA/2J and age-matched D2-Gpnmb + mice. b Representative images from immunohistochemical analyses for AKAP1 (green) and TUJ1 (red) in the retina of D2-Gpnmb + and glaucomatous DBA/2J mice. Arrowheads indicate accumulation of AKAP1 co-labeled with TUJ1 in RGC somas and arrows indicate TUJ1-labeled axon bundles. Note that glaucomatous RGCs showed a decrease in AKAP1 protein expression. Blue color indicates nucleus. c Quantitative analysis for fluorescent intensity showed a significant decrease in AKAP1 immunoreactivity in the retina of glaucomatous DBA/2J mice. 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 n = 10 (c); *P < 0.05 and ***P < 0.001 (two-tailed unpaired Student’s t test). Scale bar: 20 μm.

Fig. 2

Fig. 2. CaN-mediated dephosphorylation of Drp1 at S637 in glaucomatous retina.

a Western blot analyses for CaN in the retinas of 10-month-old glaucomatous DBA/2J and age-matched D2-Gpnmb + mice. b Representative images from immunohistochemical analyses for CaN (green, arrowheads) co-labeled with RBPMS (red, arrowheads) in RGCs. Note that glaucomatous RGCs showed increases in CaN protein expression. Blue color indicates nucleus. c Quantitative analysis for fluorescent intensity showed a significant increase in CaN immunoreactivity in the retina of glaucomatous DBA/2J mice. d Western blot analyses for total Drp1 and phospho-Drp1 Ser637 in the retinas of glaucomatous DBA/2J and age-matched D2-Gpnmb + mice. e Representative images from immunohistochemical analyses for total Drp1 (green, arrowheads) co-labeled with TUJ1 (red, arrowheads) in RGCs. Note that glaucomatous RGCs showed increases in total Drp1 protein expression. Blue color indicates nucleus. f Quantitative analysis for fluorescent intensity showed a significant increase in Drp1 immunoreactivity in the retina of glaucomatous DBA/2J mice. 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, d) and n = 10 (c, f); **P < 0.01 and ***P < 0.001 (two-tailed unpaired Student’s t test). Scale bar: 20 μm.

Fig. 3

Fig. 3. CaN expression in AKAP1−/− retina.

a Quantitative RT-PCR analysis for AKAP1 in the retinas of AKAP1−/− and WT mice. b Western blot analysis for CaN in the retinas of AKAP1−/− and WT mice. c Representative images from immunohistochemical analyses for CaN (green, arrowheads) co-labeled with RBPMS (red, arrowheads) in RGCs. Note that glaucomatous RGCs showed increases in CaN protein expression in the GCL and INL. Blue color indicates nucleus. d Quantitative analysis for fluorescent intensity showed a significant increase in CaN immunoreactivity in the retina of AKAP1−/− mice. 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 4 (a, b) and n = 10 (d); **P < 0.01 and ***P < 0.001 (two-tailed unpaired Student’s t test). Scale bar: 20 μm.

Fig. 4

Fig. 4. Phosphorylation of AMPK at Thr172 and Drp1 at Ser637 in AKAP1−/− retina.

a Western blot analysis for AMPK and phospho-AMPK Thr172 in the retinas of AKAP1−/− and WT mice. b Western blot analysis for total Drp1, phospho-Drp1 Ser637 and phospho-Drp1 Ser616 in the retinas of AKAP1−/− and WT mice. c 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 in total Drp1 protein expression in RGCs. Blue color indicates nucleus. d Quantitative analysis for fluorescent intensity showed a significant increase in Drp1 immunoreactivity in the retina of AKAP1−/− mice. 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) and n = 10 (d); *P < 0.05 (two-tailed unpaired Student’s t test). Scale bar: 20 μm.

Fig. 5

Fig. 5. Mitochondrial fission in AKAP1−/− RGC somas.

a Quantitative RT-PCR analyses for OPA1 and Mfn1 and Western blot analysis for total OPA1 (L-OPA1 & S-OPA1), and Mfn1 and 2 in the retinas of AKAP1−/− and WT mice. b–d EM and EM tomography analyses for mitochondrial fission and loss in AKAP1−/− RGC somas. 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 density 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 density of AKAP1−/− RGC somas. d Top view of the surface-rendered volume showing three 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 density 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 unpaired Student’s t test). Scale bars: 200 nm.

Fig. 6

Fig. 6. Mitophagosome formation in AKAP1−/− RGC somas.

a Western blot analyses for LC3 and p62 in the retinas of AKAP1−/− and WT mice. b Representative images from immunohistochemical analyses for LC3 (green) co-labeled with TUJ1 (red) in RGCs and their axons. Note that LC3 immunoreactivity was localized in RGC somas (arrowheads) and their axons (arrows) in WT and AKAP1−/− 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. Scale bar, 20 μm. c–j EM tomography and 3D reconstruction analysis. c A 1.6 nm thick slice through the tomographic volume of the RGC showing an autophagosome with a degenerating mitochondrial fragment inside. A mitochondrion is nearby with a strand of ER positioned in between the autophagosome and mitochondrion. ER is typically involved with mitochondrial fission. The double membrane of the autophagosome is only partially visualized and so is indicated by white arrowheads on opposite sides. For comparison, the double membrane of the mitochondrial periphery is indicated by black arrowheads on opposite sides to emphasize that the spacing between individual autophagosomal membranes is about the same as the spacing between individual mitochondrial peripheral membranes, as it should be. Labels: A, autophagosome; M, mitochondrion; PM, RGC plasma membrane; ER, endoplasmic reticulum strand. Scale = 200 nm. d Another slice through the RGC volume showing a small stretch of clearly separated components of the autophagosomal double membrane (white arrowheads). For comparison, the spacing of the mitochondrial double membrane is indicated by the black arrowheads. e A third slice through the RGC volume that best shows the double membrane of the mitochondrial fragment (white arrowheads) inside the autophagosome. Perhaps the reason that less than half the mitochondrial double membrane is seen is because it was being degraded at the time the tissue was fixed. The spacing between the individual membranes of the fragment is the same as the spacing between the individual membranes of the mitochondrion (black arrowheads). f Side view with the mitophagosome membrane made transparent to see the engulfed mitochondrion. g Side view with the outer mitochondrial membrane made transparent to see the two fragments of the inner boundary membrane (IBM) and the one crista that remain. h Top view showing the crista and right-hand portion of the IBM inside the mitochondrion, which is inside the mitophagosome. i, j 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 unpaired Student’s t test). Scale bar: 500 nm.

Fig. 7

Fig. 7. OXPHOS dysfunction and oxidative stress in AKAP1−/− retinas.

a Western blot analyses for OXPHOS Cxs in the retinas of AKAP1−/− and WT mice. b Western blot analyses for SOD2 in the retinas of AKAP1−/− and WT mice. c Representative images from immunohistochemical analyses for SOD2 (green, arrowheads) co-labeled with Brn3a (red, arrowheads) in the retina. Note that the AKAP1−/− retina shows increases in SOD2 protein expression in the inner retinal layer compared with WT retina. Blue color indicates nucleus. d Quantitative analysis for fluorescent intensity showed a significant increase in SOD2 immunoreactivity in the retina of AKAP1−/− mice. 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), n = 4 (b) and n = 10 (d). *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed unpaired Student’s t test). Scale bar: 20 μm.

Fig. 8

Fig. 8. Akt inactivation and Bim/Bax activation in AKAP1−/− retinas.

a Western blot analyses for Akt, phospho-Akt Ser473 and phospho-Akt Thr308 in the retinas of AKAP1−/− and WT mice. b Representative images from immunohistochemical analyses for Akt (green, arrowheads) co-labeled with Brn3a (red, arrowheads) in the retina. Note that AKAP1−/− retinas show increases in Akt protein expression in the INL and GCL compared with WT retinas. Blue color indicates nucleus. c Quantitative analysis for fluorescent intensity showed a significant increase in Akt immunoreactivity in the retina of AKAP1−/− mice. d Western blot analyses for Bim and Bax in the retinas of AKAP1−/− and WT mice. e Representative images from immunohistochemical analyses for Bax (green, arrowheads) co-labeled with RBPMS (red, arrowheads) in the retina. Note that AKAP1−/− retinas show increases in Bax protein expression in the GCL compared with WT retinas. Blue color indicates nucleus. f Quantitative analysis for fluorescent intensity showed a significant increase in Bax immunoreactivity in the retina of AKAP1−/− mice. 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 4 (a, d) and n = 10 (c, f). *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed unpaired Student’s t test). Scale bar: 20 μm.

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