Neuronal ATF4 contributes to 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. (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). (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 ISR-activating kinase PERK. 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).

The PERK-activated ISR is a prominent contributor to the transcriptional response to optic nerve injury, primarily acting through 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 wildtype and cKO retinae after injury (p<0.01 and FDR<0.2) were classified as PERK-, ATF4-, or CHOP-regulated. (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. 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).

PERK regulates transcriptional changes through canonical 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) wildtype 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 wildtype or 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 wildtype injury conditions between these two independent studies. Names of representative ATF4-dependent transcripts are burnt orange text and representative 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).

Cell-autonomous expression of ATF4- and CHOP-dependent transcripts by 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 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. (H) scRNA-seq data set (Tran et al., 2019) reveals RGC-autonomous activation of canonical 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 for selected ATF4- and CHOP-dependent transcripts and the RGC marker gene Rbpms that were probed by RNAScope of fresh-frozen retinal cyrosections. (D-E) Multiplex RNAScope across wildtype (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.

PERK-ATF4 contributes to 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 wildtype 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 regeneration enabled 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).

Intravitreal AAV2-hSyn1-Cre results in recombination of floxxed alleles in the inner retina.

(A) Whole-mount retina of Ai14 LSL.tdTomato Cre reporter mice immunostained for two markers of RGCs (TuJ1, Brn3a). (B) Cryosections of AAV2-hSyn1-Cre-injuected Ai14 retinas reveal recombination in cells of the ganglion cell layer (GCL) and inner nuclear layer (INL). (C-D) Multiplex RNAScope of fresh-frozen retinal cryosections confirms injury-induced upregulation of Atf4 and Ddit3/CHOP mRNA in the Rbpms+ RGCs of the GCL that is absent in ATF4 cKO and CHOP cKO, respectively. As predicted by whole retina RNA-seq, expression of the RGC marker Rbpms is reduced after injury, especially in wildtype (WT) and 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 labelled with DAPI.

Prominent representation in RGCs of injury-responsive transcripts detected by whole retina transcriptomics.

(A-C) Scatterplots showing expression level (transcripts per cell x % expressing cells) of the 282 injury-responsive genes in 3 days post ONC bulk RNA-seq identified in this study in RGC scRNA-seq dataset from Tran et al. (2019) at 1, 2, and 4 days post crush. 271/282 injury-responsive genes were detected the scRNA-seq dataset and showed a general correspondence in trend 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. (D) Multiplex fluorescent in situ hybridization (RNAScope) of fresh-frozen retinal cryosections confirms that whole retina transcriptomics can even report RGC-autonomous expression of transcripts like Avil, which exhibits substantial injury-induced upregulation in only a subset of Rbpms+ RGCs in an ATF4- and CHOP-dependent manner. As predicted by whole retina RNA-seq, expression of the RGC marker Rbpms is reduced after injury, especially in wildtype (WT) and CHOP cKO conditions. (E-F) Similar correlations and slopes upon re-analysis of linear regression between (E) PERK- and ATF4-dependent transcripts and (F) 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) suggests that these relationships are driven primarily by RGC-autonomous expression changes.

Strong concordance between this and an independent study regarding the RGC-autonomous upregulation of numerous known ATF4 target genes following optic nerve crush, but minimal concordance regarding ATF4- and CHOP-dependence.

(A) Strong 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 CHOP target genes (gold text), with greater sensitivity afforded by enriching for RGCs demonstrated by Slope>1. (B-C) 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 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, 2023) and corresponding updates to GEO:GSE184547 and 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).