1. Neuroscience

Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by ATF4 following optic nerve injury

  1. Preethi Somasundaram
  2. Madeline M Farley
  3. Melissa A Rudy
  4. Katya Sigal
  5. Andoni I Asencor
  6. David G Stefanoff
  7. Malay Shah
  8. Puneetha Goli
  9. Jenny Heo
  10. Shufang Wang
  11. Nicholas M Tran
  12. Trent A Watkins  Is a corresponding author
  1. Departments of Neurosurgery, Baylor College of Medicine, United States
  2. Division of Neuroimmunology and Glial Biology, Department of Neurology, University of California, San Francisco, United States
  3. Mol. and Human Genetics, Baylor College of Medicine, United States
https://doi.org/10.7554/eLife.87528.3
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Cite this article
  1. Preethi Somasundaram
  2. Madeline M Farley
  3. Melissa A Rudy
  4. Katya Sigal
  5. Andoni I Asencor
  6. David G Stefanoff
  7. Malay Shah
  8. Puneetha Goli
  9. Jenny Heo
  10. Shufang Wang
  11. Nicholas M Tran
  12. Trent A Watkins
(2026)
Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by ATF4 following optic nerve injury
eLife 12:RP87528.
https://doi.org/10.7554/eLife.87528.3
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5 figures, 1 table and 4 additional files

Figures

Figure 1 with 4 supplements
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Neuronal activating transcription factor-4 (ATF4) contributes to retinal ganglion cell (RGC) neurodegeneration after optic nerve crush.

(A) Neuronal knockout of ATF4, but not CHOP (Ddit3), by intravitreal AAV2-hSyn1-mTagBFP2-ires-Cre in conditional knockout (cKO) mice improves survival of RGCs 14 days after optic nerve crush, as assessed by immunostaining for the pan-RGC marker RBPMS (n=3-4 mice/condition). (B) Neuronal knockout of ATF4 similarly improves survival of RGCs when assessed by the robust nuclear marker of injured RGCs, phospho-c-Jun (p-c-Jun) (n=5-7 mice/condition). (C, D) Comparable levels of neuroprotection are conferred by ATF4 cKO and (C) ATF4/CHOP double cKO (dcKO), or (D) Eif2ak3 cKO, which results in deletion of the Integrated Stress Response (ISR)-activating kinase PERK (n=5-7 mice/condition). Error bars are SEM. Statistical analysis: one-way ANOVA with Tukey’s multiple comparison with a single pooled variance (*p<0.05; ***p<0.001; ****p<0.0001).

Figure 1—source data 1

Four worksheets, one per figure panel, reporting calculated RGC survival values plotted in Figure 1.

https://cdn.elifesciences.org/articles/87528/elife-87528-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
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Intravitreal AAV2-hSyn1-Cre results in recombination of floxed alleles in the inner retina.

Whole-mount retina of Ai14 LSL.tdTomato Cre reporter mice immunostained for two markers of retinal ganglion cells (RGCs) (TuJ1, Brn3a). Scale bar = 50 µm.

Figure 1—figure supplement 2
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Intravitreal AAV2-hSyn1-Cre results in recombination of floxed alleles in the inner retina.

Cryosections of AAV2-hSyn1-Cre-injected Ai14 retinas reveal recombination (expression of tdTomato) in cells of the ganglion cell layer (GCL) and inner nuclear layer (INL). Scale bar = 50 µm.

Figure 1—figure supplement 3
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Intravitreal AAV2-hSyn1-Cre results in loss of Atf4 mRNA in the retinal ganglion cell layer of activating transcription factor-4 (ATF4) conditional knockout mice.

Multiplex RNAScope of fresh-frozen retinal cryosections confirms injury-induced upregulation of Atf4 mRNA in the Rbpms+ retinal ganglion cells (RGCs) of the ganglion cell layer (GCL) that is absent in ATF4 conditional knockout (cKO). As predicted by whole retina RNA-seq, expression of the RGC marker Rbpms is reduced after injury, especially in wild-type (WT) and C/EBP homologous protein (CHOP) cKO conditions. Though reduced in CHOP cKO, as predicted by whole retina RNA-seq, injury-induced Ecel1 aids in the identification of the GCL. Nuclei are labeled with DAPI. Scale bar = 50 µm.

Figure 1—figure supplement 4
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Intravitreal AAV2-hSyn1-Cre results in loss of Ddit3/CHOP mRNA in the retinal ganglion cell layer of C/EBP homologous protein (CHOP) conditional knockout mice.

Multiplex RNAScope of fresh-frozen retinal cryosections confirms injury-induced upregulation of Ddit3/CHOP mRNA in the ganglion cell layer (GCL) that is absent in CHOP conditional knockout (cKO). Though reduced in CHOP cKO, as predicted by whole retina RNA-seq, injury-induced Ecel1 aids in the identification of the GCL. Nuclei are labeled with DAPI. Scale bar = 50 µm.

Figure 2
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The PERK-activated Integrated Stress Response (ISR) is a prominent contributor to the transcriptional response to optic nerve injury, primarily acting through activating transcription factor-4 (ATF4).

(A) Venn diagrams of 282 transcripts exhibiting significant differences by RNA-seq between uninjured retinae and 3 days after optic nerve crush (FDR <0.05). Amongst those, transcripts that exhibit differences between wild-type and conditional knockout (cKO) retinae after injury (p<0.01 and FDR <0.2) were classified as PERK-, ATF4-, or CHOP-regulated (n=4-5 retinae/condition). (B–D) Linear regression analyses comparing the impact of neuronal PERK deletion (Eif2ak3 cKO), ATF4 deletion (Atf4 cKO), and CHOP deletion (Ddit3 cKO) on the same 282 injury-responsive transcripts (n=4-5 retinae/condition). Names of representative ATF4-dependent transcripts are burnt orange text and representative CHOP-dependent transcripts are gold text. Significant changes are indicated by colored dots (FDR <0.05).

Figure 3
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PERK regulates transcriptional changes through canonical activating transcription factor-4 (ATF4) target genes and influence on c-Jun-regulated programs.

(A, B) Volcano plots of Ingenuity Pathway Analysis (IPA) implicate ATF4 and c-Jun as potential Upstream Transcriptional Regulators of injury-induced expression changes in (A) wild-type retina, and (B) those whose activity after injury is reduced by neuronal PERK deletion. Red, orange, blue, and light blue dots indicate Z>2.5, p<10–13; Z>1.5, p<10–10; Z<−2.5, p<10–13; and Z<−1.5, p<10–10, respectively. (C) Heat map of RNA-seq data, showing known and putative ATF4 target genes after injury in wild-type or conditional knockout (cKO) retinae. (D–F) Linear regression analyses comparing current whole retina RNA-seq cKO data to published data from a similarly designed experiment (Syc-Mazurek et al., 2022). Data from that independent study is indicated on the y-axis by &. (D) Strong correlation among 282 injury-responsive transcripts in the respective wild-type injury conditions between these two independent studies. Names of representative ATF4-dependent transcripts are burnt orange text and representative C/EBP homologous protein (CHOP)-dependent transcripts are gold text. (E) Significant correlations between the impacts of neuronal PERK conditional knockout (this study) and deletion of c-Jun from the majority of neural retina& (p<0.001). (F) Cross-study comparison of CHOP (Ddit3) cKO (this study) and CHOP (Ddit3) KO& shows strong correlation between the few CHOP-dependent injury-responsive transcripts (red dots).

Figure 4 with 6 supplements
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Cell-autonomous expression of activating transcription factor-4 (ATF4)- and C/EBP homologous protein (CHOP)-dependent transcripts by retinal ganglion cells (RGCs).

(A) Volcano plot of retinal expression data three days after optic nerve crush (this study) for 597 transcripts that are significantly altered autonomously in RGCs at day 2 or day 4 after crush, as determined by single-cell RNA-seq (scRNA-seq) (Tran et al., 2019). 287 of these transcripts are detected at FDR <0.05 (red = upregulated, or blue = downregulated) or |log2FC|>0.25 (light red = upregulated, or light blue = downregulated) in whole retina. Only three transcripts (yellow dots) were significantly regulated in whole retina in the discordant direction from scRNA-seq findings. (B) scRNA-seq data set (Tran et al., 2019) reveals RGC-autonomous activation of canonical Integrated Stress Response (ISR) transcripts. Dot plot at 2 and 4 days after injury, showing both pan-RGC and type-specific upregulation of transcripts demonstrated by whole retina RNA-seq to be ATF4- and/or CHOP-dependent. Some transcripts exhibiting low, non-uniform, injury induction in RGCs – Avil, Gm29374, Cdsn, and Fibin – were amongst those that appear to exhibit potential CHOP-dependence but failed to reach threshold for inclusion in either retinal injury-dependence, CHOP-dependence, or scRNA-seq pseudo-bulk analyses. (C–E) Fluorescent in situ hybridization (RNAScope) of ATF4- and CHOP-dependent transcripts. (C) Box-and-whisker plots of whole retina transcriptomics (n=4-5 retinae per condition) for selected ATF4- and CHOP-dependent transcripts and the RGC marker gene Rbpms that were probed by RNAScope of fresh-frozen retinal cryosections. (D, E) Multiplex RNAScope across wild-type (WT) uninjured (‘uninj’) and 3 days post-crush (‘3d’) for three genotypes (WT, ATF4 cKO, and CHOP cKO) demonstrating prominent expression by Rbpms+ RGCs of injury-induced transcripts that are reduced by knockout of ATF4 (Atf3, Chac1) or by knockout of either ATF4 or CHOP (Ecel1, Avil), concordant with RNA-seq findings. Nuclei are labeled with DAPI. Scale bars = 50 µm.

Figure 4—figure supplement 1
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Prominent representation in retinal ganglion cells (RGCs) of injury-responsive transcripts detected by whole retina transcriptomics.

Scatterplots showing RGC expression (transcripts per cell × % expressing cells) in an independent single-cell RNA-seq (scRNA-seq) dataset (Tran et al., 2019) of genes identified in this study as injury-responsive by bulk retina RNA-seq. 271/282 injury-responsive genes were detected in the scRNA-seq dataset and showed a general correspondence at each time point. This was most clear at 4 days post-crush (4dpc), in which 232/271 detected genes showed a>0.5 log2 fold change difference from control in the same direction as this study. Genes exhibiting >1 log2 change are labeled. This demonstrates that our expression differences from bulk-RNA seq identify RGC-specific expression changes and agree with previous pseudo-bulk analysis of RGC scRNA-seq data from similar time points.

Figure 4—figure supplement 2
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Detection of Avil transcript by whole retina RNA-seq reflects injury-induced expression in a subset of retinal ganglion cells (RGCs).

Multiplex fluorescent in situ hybridization (RNAScope) of fresh-frozen retinal cryosections confirms that whole retina transcriptomics reports RGC-autonomous expression of Avil, which exhibits substantial injury-induced upregulation in only a subset of Rbpms+RGCs in an activating transcription factor-4 (ATF4)- and C/EBP homologous protein (CHOP)-dependent manner. As predicted by whole retina RNA-seq, expression of the RGC marker Rbpms is reduced after injury, especially in wild-type (WT) and CHOP conditional knockout (cKO) conditions. Scale bar = 50 µm.

Figure 4—figure supplement 3
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Linear regressions of PERK-, activating transcription factor-4 (ATF4)-, and c-Jun-dependent transcripts filtered by retinal ganglion cell (RGC)-autonomous injury-regulated transcripts.

Similar correlations and slopes upon re-analysis of linear regression between PERK- and ATF4-dependent transcripts or PERK- and c-Jun-dependent transcripts using 287 genes detected as injury-responsive by both whole retina RNA-seq (|log2FC >0.25|) and RGC scRNA-seq (Tran et al., 2019) suggest that these relationships are driven primarily by RGC-autonomous expression changes.

Figure 4—figure supplement 4
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Strong concordance between this and an independent study regarding the retinal ganglion cell (RGC)-autonomous upregulation of numerous known activating transcription factor-4 (ATF4) target genes following optic nerve crush.

Robust linear regression correlation among 597 injury-induced RGC-autonomous expression changes (as determined by scRNA-seq, Tran et al., 2019) that are detected in both whole retina (this study) and FACS-enriched RGCs (FDR <0.1, Tian et al., 2022) in control (no gene targeting) conditions. This suggests that both RNA-seq approaches readily detect RGC-autonomous changes in known ATF4 target genes (burnt orange text) and putative C/EBP homologous protein (CHOP) target genes (gold text), with greater sensitivity afforded by enriching for RGCs demonstrated by Slope >1. X-axis data is from the current study (whole retina conditional knockout, cKO), and Y-axis data is from Tian et al., 2022 (FACS-sorted RGCs expressing gRNAs), as reflected in that manuscript’s Corrections (Tian et al., 2024; Tian et al., 2023) and corresponding updates to GEO:GSE184547#.

Figure 4—figure supplement 5
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Minimal concordance between this and an independent study regarding the ctivating transcription factor-4 (ATF4) dependence of known ATF4 target genes following optic nerve crush.

Linear regression comparing the impacts of targeting Atf4 by conditional knockout (cKO) or gRNA on 597 transcripts that are significantly altered in retinal ganglion cells (RGCs) at day 2 or day 4 after crush, as determined by scRNA-seq (Tran et al., 2019). X-axis data is from the current study (whole retina cKO), and Y-axis data is from Tian et al., 2022 (FACS-sorted RGCs expressing gRNAs), as reflected in that manuscript’s Corrections (Tian et al., 2024; Tian et al., 2023) and corresponding updates to GEO:GSE190667#. gRNAs significantly modulate transcripts unaffected by cKO (green dots), but do not significantly impact the expression of many transcripts impacted by cKO (blue dots).

Figure 4—figure supplement 6
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Minimal concordance between this and an independent study regarding the C/EBP homologous protein (CHOP) dependence of retinal ganglion cell (RGC)-autonomous injury-induced changes following optic nerve crush.

Linear regression comparing the impacts of targeting (B) Atf4 or (C) Ddit3/CHOP by cKO or gRNA on 597 transcripts that are significantly altered in RGCs at day 2 or day 4 after crush, as determined by scRNA-seq (Tran et al., 2019). X-axis data is from the current study (whole retina conditional knockout, cKO), and Y-axis data is from Tian et al., 2022 (FACS-sorted RGCs expressing gRNAs), as reflected in that manuscript’s Corrections (Tian et al., 2024; Tian et al., 2023) and corresponding updates to GEO:GSE190667#. gRNAs significantly modulate transcripts unaffected by cKO (green dots), but do not significantly impact the expression of transcripts impacted by cKO (blue dots).

Figure 5
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PERK-ATF4 contributes to retinal ganglion cell (RGC) axon regenerative potential after optic nerve crush.

(A, B) Heat maps of select injury-responsive transcripts revealed by retinal RNA-seq three days after optic nerve crush in wild-type and cKO mice, focusing on (A) genes implicated in axon regeneration, and (B) the maintenance of mature and subtype-specific RGC phenotypes. (C, D) Double conditional knockout (dcKO) of neuronal PERK (Eif2ak3; n=4) or ATF4 (Atf4; n=7) reduces RGC axon regeneration compared to conditional knockout (cKO) of the tumor suppressor PTEN alone (n=8). Error bars are SEM. Statistical analysis: two-way ANOVA with Sidak’s multiple comparisons test (**p<0.01).

Figure 5—source data 1

Measurements of axons growing 0.75-mm and 1.5-mm past the injury site 15 days after optic nerve crush, graphed in Figure 5D.

https://cdn.elifesciences.org/articles/87528/elife-87528-fig5-data1-v1.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)C57BL/6-Atf4tm1.1Cmad/J (Atf4 cKO /Atf4fl/fl)Christopher Adams, University of Iowa (Ebert et al., 2012)RRID:IMSR_JAX:033380 Also available Jackson Laboratory
Genetic reagent (Mus musculus)B6.Cg-Ddit3tm1.1Irt/J (Chop cKO / Ddit3fl/fl)Jackson Laboratory (Zhou et al., 2015)RRID:IMSR_JAX:030816
Genetic reagent (Mus musculus)Eif2ak3tm1.2Drc/J (Perk cKO / Eif2ak3fl/fl)Jackson Laboratory (Zhang et al., 2002)RRID:IMSR_JAX:023066
Genetic reagent (Mus musculus)B6.129S4-Ptentm1Hwu/J (Pten cKO / Ptenfl/fl)Jackson Laboratory (Lesche et al., 2002)RRID:IMSR_JAX:006440
Genetic reagent (Mus musculus)B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14 Rosa26-LSL-tdtomato)Jackson Laboratory (Madisen et al., 2010)RRID:IMSR_JAX:007914
AntibodyPhospho-c-Jun (Ser73) (D47G9) Rabbit Monoclonal #3270Cell Signaling TechnologyRRID:AB_21295751:800
AntibodyAnti-RBPMS Guinea Pig polyclonal #1832-RBPMSPhospho SolutionsRRID:AB_24922261:250
AntibodyPurified anti-Tubulin β3 (TUBB3) [TUJ1]; Mouse Monoclonal #801202BiolegendRRID:AB_100634081:1250
AntibodyNeuN (D4G4O) Rabbit Monoclonal #24307Cell Signaling TechnologyRRID:AB_26511401:800
AntibodyAlexa Fluor 647 Anti-BRN3A Rabbit Monoclonal [EPR23257-285] ab300744abcamRRID:AB_29160381:100
Sequence-based reagentRNAScope probe Mm-Rbpms-C3Advanced Cell DiagnosticsCat. No. 527231-C3
Sequence-based reagentRNAScope probe Mm-Ecel1-C2Advanced Cell DiagnosticsCat. No. 475331-C2
Sequence-based reagentRNAScope probe Mm-Ecel1-C3Advanced Cell DiagnosticsCat. No. 475331-C3
Sequence-based reagentRNAScope probe Mm-Atf4Advanced Cell DiagnosticsCat. No. 405101
Sequence-based reagentRNAScope probe Mm-Chac1Advanced Cell DiagnosticsCat. No. 514501
Sequence-based reagentRNAScope probe Mm-Avil-C2Advanced Cell DiagnosticsCat. No. 498531-C2
Sequence-based reagentRNAScope probe Mm-Atf3Advanced Cell DiagnosticsCat. No. 426891
Sequence-based reagentRNAScope probe Mm-Ddit3Advanced Cell DiagnosticsCat. No. 317661

Additional files

Supplementary file 1

RNA-seq of whole retina 3 days after optic nerve crush across four different conditions: wild-type injured compared to wild-type uninjured; ATF4 cKO injured compared to wild-type injured; PERK cKO injured compared to wild-type injured; CHOP cKO injured compared to wild-type injured.

https://cdn.elifesciences.org/articles/87528/elife-87528-supp1-v1.xlsx
Supplementary file 2

Results of the Upstream Regulator tool of Ingenuity Pathway Analysis (IPA) for transcripts significantly regulated by injury in wild-type retina 3 days after optic nerve crush.

https://cdn.elifesciences.org/articles/87528/elife-87528-supp2-v1.xlsx
Supplementary file 3

Results of the Upstream Regulator tool of Ingenuity Pathway Analysis (IPA) for differentially expressed transcripts between PERK cKO and wild-type retinas 3 days after optic nerve crush.

https://cdn.elifesciences.org/articles/87528/elife-87528-supp3-v1.xlsx
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  1. Preethi Somasundaram
  2. Madeline M Farley
  3. Melissa A Rudy
  4. Katya Sigal
  5. Andoni I Asencor
  6. David G Stefanoff
  7. Malay Shah
  8. Puneetha Goli
  9. Jenny Heo
  10. Shufang Wang
  11. Nicholas M Tran
  12. Trent A Watkins
(2026)
Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by ATF4 following optic nerve injury
eLife 12:RP87528.
https://doi.org/10.7554/eLife.87528.3