Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by Atf4 following optic nerve injury

Reviewer #1 (Public Review):

Somasundaram and colleagues explore the role of transcription factors in retinal ganglion cell (RGC) death and axonal regeneration after a disease relevant insult (mechanical axonal injury). The work significantly extends our knowledge of the role of MAPK and integrated stress response (ISR) in controlling RGC fate after injury. Specifically, the manuscript shows that after axonal injury PERK-activated ISR acts through Atf4 to drive a prodeath transcriptional response in RGCs, in part by crosstalk with the prodeath JUN transcriptional program. Also, and perhaps most interesting, the work shows that PERK-ATF4 pathway activation is pro-regenerative for RGC axons. A major plus of the manuscript is that many new RNA-seq datasets are generated that describe the major prodegenerative and proregenerative gene networks altered after axonal injury.

A limitation of the study is that it does not directly compare the effect of inhibiting the PERK-ATF4 pathway with inhibiting JUN and/or JUN-CHOP double deficient animals. It would also be useful, for the cell survival experiments shown in Figure 1, to examine a longer time point than 14 days to understand the long-term consequence of manipulating the PERK-ATF4 pathway.

Reviewer #2 (Public Review):

This manuscript investigates the role of Perk (Protein kinase RNA-like endoplasmic reticulum kinase) and Atf4 (Activating Transcription Factor-4) in neurodegenerative and regenerative responses following optic nerve injury. The authors employed conditional knockout mice to examine the impact of the Perk/Atf4 pathway on transcriptional responses, with a particular focus on canonical Atf4 target genes and the involvement of C/ebp homologous protein (Chop).

The study demonstrates that Perk primarily operates through Atf4 to stimulate both pro-apoptotic and pro-regenerative responses after optic nerve injury. This Perk/Atf4-dependent response encompasses canonical Atf4 target genes and limited contributions from Chop, exhibiting overlap with c-Jun-dependent transcription. Consequently, the Perk/Atf4 pathway appears crucial for coordinating neurodegenerative and regenerative responses to central nervous system (CNS) axon injury. Additionally, the authors observed that neuronal knockout of Atf4 mimics the neuroprotection resulting from Perk deficiency. Moreover, Perk or Atf4 knockout hinders optic axon regeneration facilitated by the deletion of the tumor suppressor Pten.

These findings contrast with the transcriptional and functional outcomes reported for CRISPR targeting of Atf4 or Chop, revealing a vital role for the Perk/Atf4 pathway in orchestrating neurodegenerative and regenerative responses to CNS axon injury.

However, the main concern is the overall data quality, which appears to be suboptimal. The transfection efficiency of AAV2-hSyn1-mTagBFP2-ires-Cre used in this study does not seem highly effective, as evidenced by the data presented in Supplementary Figure 1. The manuscript also contains several inconsistencies and a mix of methods in data collection, analysis, and interpretation, such as the labeling and quantification of RGCs and the combination of bulk and single-cell sequencing results.

Despite these limitations, the study offers valuable insights into the role of the Perk/Atf4 pathway in determining neuronal fate after axon injury, emphasizing the significance of understanding the molecular mechanisms that govern neuronal survival and regeneration. This knowledge could potentially inform the development of targeted therapies to promote neuroprotection and CNS repair following injury.