Remarks for the authors: Tribble and colleagues describe early changes of microglial genes in optic nerve head of DBA/2J mice. These changes included down-regulation or inhibition of homeostatic function of microglia. They also found upregulation of metabolism-related genes and changes in mitochondrial genes are early features of microglia in the pre-glaucomatous ONH of D2 mouse. They further demonstrated that radiation therapy at 10 weeks of age restores microglial genes at 9 months old. Overall this study contains compelling and suggestive dataset for understanding novel mechanism of microglia at the disease onset of glaucoma. I believe the paper will be well read and well cited.

I have a few concerns that need to be addressed and/or discussed.

Major points 1. The biggest concern is how elevated IOP causes microglial changes at pre-glaucomatous ONH. Are there any known mechanisms that IOP-mediated mechanical stress causes down-regulation or inhibition of microglial functions such as phagocytosis and inflammation?

1. The authors found metabolic changes in microglia in pre-glaucomatous ONH of D2 mice. Their previous study (Williams et al. Science 2017) showed that supplementation of NAD+ precursor blocked development of glaucoma. Can such supplementation restore microglial function?

2. Radiation was administered to D2 mice at 10 weeks of age at which no IOP elevation was induced. Because the main cause triggering glaucoma is an elevated IOP after 6 months old, microglial phenotype at this time point (i.e. 10 weeks) should be normal. Why radiation is effective for regulating microglial function?

3. Why the effect of radiation is such long-lasting?

4. Radiation did not reduce IOP but restored microglial function. How radiation induces such changes in microglia? Is there any molecular mechanism mediating this response?

5. In addition to expression profile, histological data would be helpful for microglial changes. Does microglia show more ramified morphology, and locate close to the optic nerve or blood vessels?

6. Previous study by the authors (Howell et al. JCI 2011) has demonstrated that expression of endothelin 2 in ONH microglia are early feature of these cells. Does microglia dominantly express endothelin 2 than monocytes in the present study?

Minor points 1. What is the sex of mice used in this study? Because microglial function is highly different by sex of animal, it should be noted in the Methods section. 2. Their previous study (Williams et al. Mol Neurodegener 2019) has shown that blockade of microglial CD11b suppressed extravasation of monocytes, indicating that microglia is key player controlling tissue inflammation by monocytes and ONH damages at later time point. It is better to discuss how microglia control blood-retina barrier. For example, microglial P2Y12 receptor mediates closure of the injured blood-brain barrier (Lou et al. PNAS 2016). Supplemental data in the present study also suggests that D2 microglia at early stages (S1-S3) express lower P2ry12 gene.

1. In addition, the authors demonstrated down-regulation of Ccl5 expression in microglia. Recent study has shown that CCR5 induces microglial movement toward blood vessel in response to BBB breakdown (Haruwaka et al. Nat Com 2019). This response associates with Claudin-5 expression in microglia and support barrier function by endothelial cells. These reports should be cited and discussed.