BAX-Depleted Retinal Ganglion Cells Survive and Become Quiescent Following Optic Nerve Damage

  • Ryan J. Donahue
  • Margaret E. Maes
  • Joshua A. Grosser
  • Robert W. NickellsEmail author


Removal of the Bax gene from mice completely protects the somas of retinal ganglion cells (RGCs) from apoptosis following optic nerve injury. This makes BAX a promising therapeutic target to prevent neurodegeneration. In this study, Bax+/− mice were used to test the hypothesis that lowering the quantity of BAX in RGCs would delay apoptosis following optic nerve injury. RGCs were damaged by performing optic nerve crush (ONC) and then immunostaining for phospho-cJUN, and quantitative PCR were used to monitor the status of the BAX activation mechanism in the months following injury. The apoptotic susceptibility of injured cells was directly tested by virally introducing GFP-BAX into Bax−/− RGCs after injury. The competency of quiescent RGCs to reactivate their BAX activation mechanism was tested by intravitreal injection of the JNK pathway agonist, anisomycin. Twenty-four weeks after ONC, Bax+/− mice had significantly less cell loss in their RGC layer than Bax+/+ mice 3 weeks after ONC. Bax+/− and Bax+/+ RGCs exhibited similar patterns of nuclear phospho-cJUN accumulation immediately after ONC, which persisted in Bax+/− RGCs for up to 7 weeks before abating. The transcriptional activation of BAX-activating genes was similar in Bax+/− and Bax+/+ RGCs following ONC. Intriguingly, cells deactivated their BAX activation mechanism between 7 and 12 weeks after crush. Introduction of GFP-BAX into Bax−/− cells at 4 weeks after ONC showed that these cells had a nearly normal capacity to activate this protein, but this capacity was lost 8 weeks after crush. Collectively, these data suggest that 8–12 weeks after crush, damaged cells no longer displayed increased susceptibility to BAX activation relative to their naïve counterparts. In this same timeframe, retinal glial activation and the signaling of the pro-apoptotic JNK pathway also abated. Quiescent RGCs did not show a timely reactivation of their JNK pathway following intravitreal injection with anisomycin. These findings demonstrate that lowering the quantity of BAX in RGCs is neuroprotective after acute injury. Damaged RGCs enter a quiescent state months after injury and are no longer responsive to an apoptotic stimulus. Quiescent RGCs will require rejuvenation to reacquire functionality.


BAX Retinal ganglion cells Optic nerve crush Intrinsic apoptosis cJun Glia Neuroprotection 



retinal ganglion cell


optic nerve crush


mitochondrial outer membrane


mitochondrial outer membrane permeabilization


BCL2-homology domain, 3


BCL2 associated X, apoptosis regulator


BAX activation mechanism


c-JUN N-terminal kinase




quiescent retinal ganglion cell



We would like to thank Satoshi Kinoshita at the Translational Research Initiative in Pathology (TRIP) lab at the University of Wisconsin–Madison for cutting all retinal sections analyzed in this manuscript.

Author’s Contributions

RWN conceived the study. RJD and MEM contributed intellectually to the study design. JAG imaged and counted whole-mounted retinas. MEM conducted some of the viral injections, immunofluorescence, and imaging pertaining to GFP-BAX colocalization with TOM20. RJD performed all other experiments including all surgical procedures, immunofluorescence, qPCR, and counting of whole-mounted retinas and retinal sections. RWN prepared all the figures for the manuscript and processed images for figures. RJD and RWN wrote the manuscript. All authors contributed to editing the manuscript and approved the final manuscript.

Funding Information

This work was supported by National Eye Institute grants R01 EY012223 (RWN), R01 EY030123 (RWN), T32 EY027721 (Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison), and a Vision Science Core grant P30 EY016665 (Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison), an unrestricted funding grant from Research to Prevent Blindness (Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison), the Frederick A. Davis Endowment (RWN), and the Mr. and Mrs. George Taylor Foundation (RWN).

Compliance with Ethical Standards

Ethics Approval

Adult mice were used for this study and handled according to the Association for Research in Vision and Ophthalmology statement for the use of animals in research and all experiments were approved by the Animal Care and Use Committee at the University of Wisconsin–Madison.

Competing Interests

The authors declare that they have no competing interests.

Supplementary material

12035_2019_1783_MOESM1_ESM.jpg (759 kb)
Supplemental Figure 1 All qPCR data from Bax+/+ mice after optic nerve crush. qPCR data for wild type animals 1, 3 and 7 days after optic nerve crush (ONC). Bars represent the fold change in the retinas of experimental eyes relative to the contralateral control eyes. The data for the groups collected 1 and 3 days after ONC are the average of 2 cohorts of 3 mice each, whose eyes were pooled for analysis. The data for the group collected 7 days after ONC is the average of 4 cohorts of 3 mice each, whose eyes were pooled for analysis. Error bars represent the standard deviation from the mean. P values were calculated using a one sided t test, assuming equal variance. *P < 0.05, **P < 0.01, ***P < 0.001. (JPG 758 kb)
12035_2019_1783_MOESM2_ESM.jpg (567 kb)
Supplemental Figure 2 The average number of cells labeled per field in each cohort of mice that was transduced with AAV2/2-Pgk-GFP-Bax. Bars represent the mean number of GFP-BAX positive cells labeled in each image for each group. Error bars represent the standard deviation from the mean. P values were calculated using a one-sided test, assuming equal variance between groups. Number of mice per group is as follows: 10 for Bax+/+ transduced before ONC, 5 for Bax+/− transduced before ONC, 9 for Bax−/− transduced before ONC, 7 for Bax−/− transduced 4 weeks after ONC and 7 for Bax−/− transduced 8 weeks after ONC. *P < 0.05, **P < 0.01, ***P < 0.001. (JPG 567 kb)


  1. 1.
    Howell GR, Libby RT, Jakobs TC, Smith RS, Phalan FC, Barter JW, Barbay JM, Marchant JK et al (2007) Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma. J Cell Biol 179:1523–1537PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Li Y, Schlamp CL, Nickells RW (1999) Experimental induction of retinal ganglion cell death in adult mice. Invest Ophthalmol Vis Sci 40:1004–1008PubMedPubMedCentralGoogle Scholar
  3. 3.
    Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Edlich F, Banerjee S, Suzuki M, Cleland MM, Arnoult D, Wang C, Neutzner A, Tjandra N et al (2011) Bcl-xL retrotranslocates Bax from the mitochondria into the cytosol. Cell. 145:104–116PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Levin LA, Schlamp CL, Spieldoch RL, Geszvain KM, Nickells RW (1997) Identification of the bcl-2 family of genes in the rat retina. Invest Ophthalmol Vis Sci 38:2545–2553PubMedPubMedCentralGoogle Scholar
  6. 6.
    Harder JM, Ding Q, Fernandes KA, Cherry JD, Gan L, Libby RT (2012) BCL2L1 (BCL-X) promotes survival of adult and developing retinal ganglion cells. Mol Cell Neurosci 51:53–59PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Sun YF, Yu LY, Saarma M, Timmusk T, Arumae U (2001) Neuron-specific Bcl-2 homology 3 domain-only splice variant of Bak is anti-apoptotic in neurons, but pro-apoptotic in non-neuronal cells. J Biol Chem 276:16240–16247PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Li Y, Schlamp CL, Poulsen KP, Nickells RW (2000) Bax-dependent and independent pathways of retinal ganglion cell death induced by different damaging stimuli. Exp Eye Res 71:209–213PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Libby RT, Li Y, Savinova OV, Barter J, Smith RS, Nickells RW, John SWM (2005) Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage. PLoS Genet 1:e4PubMedCentralCrossRefGoogle Scholar
  10. 10.
    Semaan SJ, Li Y, Nickells RW (2010) A single nucleotide polymorphism in the Bax gene promoter affects transcription and influences retinal ganglion cell death. ASN Neuro 2:87–101CrossRefGoogle Scholar
  11. 11.
    Maes ME, Schlamp CL, Nickells RW (2017) Bax to basics: How the BCL2 gene family controls the death of retinal ganglion cells. Prog Retin Eye Res 57:1–25PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Fernandes KA, Harder JM, Fornarola LB, Freeman RS, Clark AF, Pang I-H, John SWM, Libby RT (2012) JNK2 and JNK3 are major regulators of axonal injury-induced retinal ganglion cell death. Neurobiol Dis 46:393–401PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Fernandes KA, Harder JM, Kim J, Libby RT (2013) JUN regulates early transcriptional responses to axonal injury in retinal ganglion cells. Exp Eye Res 112:106–117PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Putcha GV, Le S, Frank S, Besirli CG, Clark K, Chu B et al (2003) JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis. Neuron. 38:899–914PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Welsbie DS, Yang Z, Ge Y, Mitchell KL, Zhou X, Martin SE, Berlinicke CA, Hackler L et al (2013) Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc Natl Acad Sci 110:4045–4050PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Watkins TA, Wang B, Huntwork-Rodriguez S, Yang J, Jiang Z, Eastham-Anderson J et al (2013) DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury. Proc Natl Acad Sci 110:4039–4044PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Syc-Mazurek SB, Fernandes KA, Wilson MP, Shrager P, Libby RT (2017) Together JUN and DDIT3 (CHOP) control retinal ganglion cell death after axonal injury. Mol Neurodegener 12:71PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Kim EM, Jung C-H, Song J-Y, Park JK, Um H-D (2018) Pro-apoptotic Bax promotes mesenchymal-epithelial transition by binding to respiratory complex-I and antagonizing the malignant actions of pro-survival Bcl-2 proteins. Cancer Lett 424:127–135PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Reyna DE, Garner TP, Lopez A, Kopp F, Choudhary GS, Sridharan A, Narayanagari SR, Mitchell K et al (2017) Direct activation of BAX by BTSA1 overcomes apoptosis resistance in acute myeloid leukemia. Cancer Cell 32:490–505PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Karbowski M, Norris KL, Cleland MM, Jeong S-Y, Youle RJ (2006) Role of Bax and Bak in mitochondrial morphogenesis. Nature. 443:658–662PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Mosinger Ogilvie J, Deckwerth TL, Knudson CM, Korsmeyer SJ (1998) Supression of developmental retinal cell death but not of photoreceptor degeneration in Bax-deficient mice. Invest Ophthalmol Vis Sci 39:1713–1720PubMedPubMedCentralGoogle Scholar
  22. 22.
    Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ (1995) Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science. 270:96–99PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Schmitt HM, Pelzel HR, Schlamp CL, Nickells RW (2014) Histone deacetylase 3 (HDAC3) plays an important role in retinal ganglion cell death after acute optic nerve injury. Mol Neurodegener 9:39PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 25:402–408PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Schlamp CL, Montgomery AD, MacNair CE, Schuart C, Willmer DJ, Nickells RW (2013) Evaluation of the percentage of ganglion cells in the ganglion cell layer of the rodent retina. Mol Vis 19:1387–1396PubMedPubMedCentralGoogle Scholar
  26. 26.
    Nadal-Nicolas FM, Jimenez-Lopez M, Sobrado-Calvo P, Nieto-Lopez L, Canovas-Martinez I, Salinas-Navarro M et al (2009) Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naïve and optic nerve–injured retinas. Invest Opthalmology Vis Sci 50:3860–3868CrossRefGoogle Scholar
  27. 27.
    Harder JM, Libby RT (2011) BBC3 (PUMA) regulates developmental apoptosis but not axonal injury induced death in the retina. Mol Neurodegener 6:50PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Harder JM, Fernandes KA, Libby RT (2012) The Bcl-2 family member BIM has multiple glaucoma-relevant functions in DBA/2J mice. Sci Rep 2:530PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Yang X, Luo C, Cai J, Pierce WM, Tezel G (2008) Phosphorylation-dependent interaction with 14-3-3 in the regulation of bad trafficking in retinal ganglion cells. Invest Ophthalmol Vis Sci 49:2483–2494PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Happo L, Strasser A, Cory S (2012) BH3-only proteins in apoptosis at a glance. J Cell Sci 125:1081–1087PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Mac Nair CE, Nickells RW (2015) Neuroinflammation in glaucoma and optic nerve damage. Prog Mol Biol Transl Sci 134:343–363PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Nickells RW, Schmitt HM, Maes ME, Schlamp CL (2017) AAV2-mediated transduction of the mouse retina after optic nerve injury. Invest Opthalmology Vis Sci 58:6091–6104CrossRefGoogle Scholar
  33. 33.
    Laderoute KR, Webster KA (1997) Hypoxia/reoxygenation stimulates Jun kinase activity through redox signaling in cardiac myocytes. Circ Res 80:336–344PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Duan X, Qiao M, Bei F, Kim I-J, He Z, Sanes JR (2015) Subtype-specific regeneration of retinal ganglion cells following axotomy: Effects of osteopontin and mTOR signaling. Neuron. 85:1244–1256PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Daniel S, Clark A, McDowell C (2018) Subtype-specific response of retinal ganglion cells to optic nerve crush. Cell Death Dis 5:7Google Scholar
  36. 36.
    Youle RJ, Van Der Bliek AM (2012) Mitochondrial fission, fusion, and stress. Science. 337:1062–1065PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Robin AY, Iyer S, Birkinshaw RW, Sandow J, Wardak A, Luo CS et al (2018) Ensemble properties of Bax determine its function. Structure Cell Press 26:1346–1359Google Scholar
  38. 38.
    Nickells RW (2010) Variations in the rheostat model of apoptosis: What studies of retinal ganglion cell death tell us about the functions of the Bcl2 family proteins. Exp Eye Res 91:2–8PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Adams JM, Cory S (2007) The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 26:1324–1337PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Valentijn AJ, Upton J-P, Bates N, Gilmore AP (2008) Bax targeting to mitochondria occurs via both tail anchor dependent and independent mechanisms. Cell Death Differ 15:1243–1254PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Semaan SJ, Nickells RW (2010) The apoptotic response in HCT116 cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein. BMC Cancer 10:554PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Maes ME, Schlamp CL, Nickells RW (2017) Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis. PLoS One 12:e0184434PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Kermer P, Klöcker N, Labes M, Thomsen S, Srinivasan A, Bähr M (1999) Activation of caspase-3 in axotomized rat retinal ganglion cells in vivo. FEBS Lett 453:361–364PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Kermer P, Klöcker N, Labes M, Bähr M (2000) Insulin-like growth factor-I protects axotomized rat retinal ganglion cells from secondary death via PI3-K-dependent Akt phosphorylation and inhibition of caspase-3 in vivo. J Neurosci 20:2–8PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Schlamp CL, Johnson EC, Li Y, Morrison JC, Nickells RW (2001) Changes in Thy1 gene expression associated with damaged retinal ganglion cells. Mol Vis 7:192–201PubMedPubMedCentralGoogle Scholar
  46. 46.
    Janssen KT, Mac Nair CE, Dietz JA, Schlamp CL, Nickells RW (2013) Nuclear atrophy of retinal ganglion cells precedes the bax-dependent stage of apoptosis. Invest Ophthalmol Vis Sci 54:1805–1815PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Pelzel HR, Schlamp CL, Waclawski M, Shaw MK, Nickells RW (2012) Silencing of Fem1cR3 gene expression in the DBA/2J mouse precedes retinal ganglion cell death and is associated with histone deacetylase activity. Invest Ophthalmol Vis Sci 53:1428–1435PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Pelzel HR, Schlamp CL, Nickells RW (2010) Histone H4 deacetylation plays a critical role in early gene silencing during neuronal apoptosis. BMC Neurosci 11:62PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Schmitt HM, Schlamp CL, Nickells RW (2016) Role of HDACs in optic nerve damage-induced nuclear atrophy of retinal ganglion cells. Neurosci Lett 625:11–15PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Yungher BJ, Ribeiro M, Park KK (2017) Regenerative responses and axon pathfinding of retinal ganglion cells in chronically injured mice. Invest Ophthalmol Vis Sci 58:1743–1750PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Ahmad K, Baig M, Gupta GK, Kamal M, Pathak N, Choi I (2016) Identification of common therapeutic targets for selected neurodegenerative disorders: an in silico approach. J Comput Sci 17:292–306CrossRefGoogle Scholar
  52. 52.
    Kudo W, Lee H-P, M a S, Zhu X, Matsuyama S, Lee H-G (2012) Inhibition of Bax protects neuronal cells from oligomeric Aβ neurotoxicity. Cell Death Dis 3:e309PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Kozin MS, Kulakova OG, Favorova OO (2018) Involvement of mitochondria in neurodegeneration in multiple sclerosis. Biochem. 83:813–830Google Scholar
  54. 54.
    Cosker KE, Pazyra-Murphy MF, Fenstermacher SJ, Segal RA (2013) Target-derived neurotrophins coordinate transcription and transport of Bclw to prevent axonal degeneration. J Neurosci 33:5195–5207PubMedPubMedCentralCrossRefGoogle Scholar
  55. 55.
    Nickells RW, Semaan SJ, Schlamp CL (2008) Involvement of the Bcl2 gene family in the signaling and control of retinal ganglion cell death. Prog Brain Res 173:423–435PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Ophthalmology and Visual SciencesUniversity of WisconsinMadisonUSA
  2. 2.Cellular and Molecular Pathology Graduate ProgramUniversity of WisconsinMadisonUSA
  3. 3.Department of Life SciencesInstitute of Science and TechnologyKlosterneuburgAustria

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