## 3 Discussion
The mitochondrial stress/malfunction might have also been reflected by the increased oxidative stress responses in the retina after the T3 treatment, manifested as an increased expression of oxidative stress genes [42] and increased labeling of oxidative stress/damage markers [37]. It seems reasonable to believe that excessive TH signaling impairs oxidative phosphorylation and bioenergetics, which may result in the accumulation of unstable molecules or free radicals and oxidative stress. Indeed, treatment with an antioxidant reduces T3-induced cone degeneration and cellular stress responses [37]. It was well characterized that excessive TH signaling induces cone death. However, the mechanism(s) behind this process remains less understood. As mentioned above, oxidative stress may play a role in the progression of cell death. The scRNAseq and IPA revealed additional cellular stress/death pathways that might be involved in T3-induced cone death. This includes the EIF2 signaling pathway and CREB signaling pathway. EIF2 signaling integrates a diverse array of stress-related signals to regulate mRNA translation, which, in turn, regulates signaling processes such as ER stress, apoptosis, amino acid metabolism, and glucose uptake. Specifically, the increased expressions of _Aft3_, _Ddit3_, and _Xiap_ suggest an involvement of EIF2-ATF3-related ER stress/apoptosis (Supplementary Table S6). CREB signaling regulates diverse cellular processes, including proliferation, survival, and differentiation. CREB signaling can be activated by cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), calmodulin kinases (e.g., CaMKII and CAMK4), and ribosomal S6 kinase (RPS6 KA1). Indeed, PKA signaling was upregulated in cones after the T3 treatment (Figure 3; Table 2). The regulation of TH signaling on CaMKII/CREB signaling was reported previously [46]. Furthermore, the IPA network analysis and the upstream regulator analysis also suggest cellular compromise and the activation of the cell death pathways. Thus, TH-induced cone death appears to involve multiple cellular events and signaling pathways. These pathways merit further investigation for their specific roles in the detrimental effects of excessive TH signaling. A comparison of DEGs in cones and rods shows a high overlap rate (70%; 36 out of 51) between the two types of photoreceptors (Supplementary Figure S4A). Among the 36 shared DEGs, 34 were altered in the same direction, and 2 were in the opposite direction (Supplementary Figure S4A; Supplementary Table S10), suggesting that the rods responded to the T3 treatment similarly to the cones. Nevertheless, T3 induces many more DEGs and canonical pathway alterations in cones compared to those induced in rods, as analyzed by the Benjamini-Hochberg adjusted \(p\)-values (Supplementary Figure S4B). T3 induces oxidative phosphorylation, EIF2 signaling, and CREB signaling, specifically in cones, whereas phototransduction, cAMP-mediated signaling, tRNA splicing, and gaseous signaling are induced in both cones and rods (Supplementary Figure S4B). Furthermore, T3 alters many more upstream regulators in cones compared with those altered in rods based on the z-scores (Supplementary Figure S4C). Of note, _Nrl_ and _Ifg_ are oppositely regulated in cones and rods (Supplementary Figure S4C). Similarly, an analysis of diseases and functions shows many more cellular events in cones compared with those in rods (Supplementary Figure S4D). Nevertheless, retinal degeneration, the degeneration of photoreceptors, organ degeneration, the neurodegeneration of sensory neurons, and neurodegeneration are among the top five upregulated cellular events in both cones and rods after T3 treatment (Supplementary Figure S4D). This work, using a mouse model of hyperhybridism, shows that excessive TH signaling suppresses phototransduction and mitochondrial bioenergetics at the transcriptomic levels and induces cellular stress/death pathways. Whether these cellular events are altered at the protein levels remains to be determined.