**4. Discussion**

Autophagy is a critical and indispensable housekeeping process in the cells of the retina. Numerous reports support the functional relevance of autophagy in specific cells of the retina and how dysregulated autophagy contributes to retinal malfunction and degeneration [**??????** ]. While acute short-term noxious stimuli (e.g., nutrient deprivation; hypoxia and endoplasmic reticulum (ER) stress; oxidative stress arising from light, lipofuscin, POS phagocytosis by RPE or mitochondrial ROS generated in retinal cells (which have a generally high metabolic rate)) can stimulate autophagy as a generally cytoprotective mechanism, it must be acknowledged that diurnal basal autophagic modulation is a critical factor in abrogating the unavoidable regular cellular damage that occurs during the basic functioning of retinal cells. An efficient regulatory molecular circuit is required in the retina, that would modulate both the intensity and duration of basal autophagic activity in specific cell types and thus meet their regular housekeeping demands [**?** ]. Previous studies have suggested the existence of autophagic rhythm in rodent heart muscles and liver, as well as kidney proximal tubules [**???** ]. In this study, we establish the existence of diurnal regulation of autophagic activity, in both the neural and vascular cells of the retina. We show evidence that certain autophagic markers like Beclin1, ATG9 and LC3 are highly expressed across the retina, while ATG7 shows preferential staining patterns and is enriched only in certain layers (**????**). The first report on diurnal rhythmicity in autophagy in the retina recorded increased autophagosome formation following outer disk shedding in rat photoreceptors [**?** ]. Our results regarding the biphasic oscillation of diurnal rhythmicity of LC3 expression agree with the findings in a report by Yao et al. 2014, who also observed two peaks of elevated autophagic activity [**?** ]. Our results in Figure **??** make a strong argumen<sup>t</sup> in favor of the influence of external stimuli such as light for the entrainment and maintenance of healthy amplitudes of oscillation of autophagic protein expression in the retina. However, we observed that even in the absence of light entrainment, the retina continued to display rhythmicity in ATG7, ATG9, LC3 and BECN, but were lower in amplitude and out of phase, suggesting that there may be an intrinsic diurnal rhythm of autophagy in the retina not dependent on light entrainment.

Our study encompasses autophagy in aging and diabetic conditions. We successfully demonstrate that perturbed autophagy is a characteristic feature of aging. It has been suggested that macroautophagy suffers a decline with aging and is replaced partly by other forms of autophagy [**?** ]. It remains to be determined if other forms of autophagy i.e., the chaperone mediated autophagy and microautophagy are also under regulation by the circadian system and, if so, whether their rhythmicity is affected in aging and disease. In addition to the above, our study reveals that the level of disruption in T1D and T2D is significant, not only in younger mice, but also in older animals (**????**). Compared to age-matched control mouse retinas, the dramatic deviations in the oscillatory patterns or the overall levels of one or more Atgs in both two- and nine-month old diabetic mouse retina could be an indication of faulty housekeeping and pathological outcomes, such as the accumulation of damaged mitochondria and ROS production [**????** ].

A limitation of this study is that it relies on immunohistochemistry to determine changes in the expression of autophagy proteins throughout the neural retina. We did not attempt to confirm our findings by assessing gene expression or protein levels using Western blot. Neither did we assess autophagic flux by determining the LC3II:LC3I ratio. The reasons for this were a combination of the large number of animals needed; regional/cell-specific changes would be masked in whole retinal preps and the non-canonical role of LC3 in retinal and immune cell phagocytosis [**?** ] could complicate the analysis of the autophagic flux. Furthermore, based on our data, there is a need to determine the mechanistic link between diurnal changes and autophagy in the neural retina. There is considerable evidence that diurnal/circadian rhythm is associated with the induction of autophagy [**? ?** ], a key regulator of autophagy; the mechanistic target of rapamycin (mTOR) is regulated by the circadian clock [**?** ] and the circadian regulation of metabolism is mediated through reciprocal signaling between the clock and metabolic regulatory networks such as autophagy [**?** ]. Recently, Ryzhikov and colleagues reported that diurnal rhythms spatially and temporarily organize autophagy [**?** ]. They reported that basal autophagy rhythms could be resolved into two antiphase clusters that were distinguished by the subcellular location of targeted proteins. Daytime autophagy was directed towards cytosolic proteins and proteosomal degradation, while nighttime autophagy was directed towards ER and mitochondrial removal. There is now a need to better understand the mechanistic control of these processes.

As mentioned above, autophagy in diabetes and related diseases has become an area of intense research, with focus on how this pathway may be targeted in synergy, with other therapeutic approaches to encourage a better clinical outcome. Our study, along with other recent reports, adds a novel extension to the mammalian diurnal rhythmicity in its relevance in regulation of biological processes. It provides a novel link between dysregulated autophagy and disrupted diurnal rhythm in the aging and diabetic retina and suggests that our analyses of myriad biological processes in the retina should be reconsidered from a diurnal perspective, in order to better comprehend age-related vision loss and disease pathology.

**Author Contributions:** Conceptualization, M.E.B., M.B.G. and J.V.B; Data curation, X.Q., S.K.M; Formal analysis, X.Q., Y.Y.; Investigation, X.Q., S.K.M., M.E.B. Resources, M.E.B., J.V.B., M.B.G.; Project administration: M.E.B., J.V.B., M.B.G. Writing—original draft preparation, X.Q., M.E.B.; Writing—review and editing, X.Q., S.K.M., Y.Y., J.V.B., M.B.G., M.E.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** M.E.B. is supported by NIH funding (EY019688, EY021626) and an unrestricted gran<sup>t</sup> from Research to Prevent Blindness. J.V.B. is supported by NIH funding (EY01EY028049, R01EY016077; R01EY025383).

**Acknowledgments:** The animal experiments were performed over a period of time when MEB, MBG, XQ and SKM were at University of Florida and then Indiana University, before moving to UAB. All data were analyzed at UAB.

**Conflicts of Interest:** The authors declare no conflict of interest.
