## 3 Discussion
Mitochondrial dynamics, especially fission and fusion, are indispensable for mitochondrial distribution and homeostasis [36, 37]. The DNM1L protein is crucially involved in this process, and its function is determined by various post-translational modifications (PTMs) such as phosphorylation, ubiquity, S-nitrosylation, and sumoylation. These modifications collectively regulate the mitochondrial fission rate [38, 39]. The best-described phosphorylation sites of DNM1L are Ser635 and Ser656. Phosphorylation of Ser635 stimulates DRP1 activity leading to increased mitochondrial fission, while phosphorylation of Ser656 inhibits fission [38, 40]. Protein kinase A (PKA)-induced phosphorylation of Ser656 reduces the GTPase activity of DNM1L and promotes DNM1L dissociation from the outer mitochondrial membrane. Dephosphorylation of Ser656 occurs via Ca\({}^{2+}\)-activated calcineurin triggering fission [41, 42]. To investigate the molecular effects of anti-DNM1L-Ab injection, total DNM1L protein expression and the phosphorylation status of Ser635 (DNM1L\({}^{\text{P635}}\)) and Ser656 (DNM1L\({}^{\text{P656}}\)) were analyzed. Western blot analysis of retinal tissues revealed increased DNM1L\({}^{\text{total}}\) abundance in the DNM1L OS group (Figure 5B), suggesting increased mitochondrial dynamics and altered localization. The function of DNM1L is versatile and is not only based on the total protein expression but is strongly influenced by various PTMs. The ratio of the phosphorylation Ser635 to Ser65 indicates higher mitochondrial fission. In turn, more DNM1L is required for fission. This would correspond to a kind of positive forward loop and could be an explanation for increased DNM1L levels. Therefore, the DNM1L total protein expression is an indicator; the phosphorylation ratio to each other (normalized to the total protein in each case) is even more decisive. However, these are considerations, and it should bear in mind that the data shown show a high standard deviation. Therefore, we do not want to overinterpret the data but consider them as indications that need to be addressed in future studies in more detail. Studies demonstrated that DNM1L could impact the localization of mitochondria or vesicles. Mitochondria are transported to sites of increased energy demand in response to DNM1L expression, thereby regulating synapse formation and axon branching [16, 43]. However, mitochondrial dynamics depend on several factors, such as PTMs. In this regard, the DNM1L\({}^{\text{P635/total}}\) expression level was similar in both study groups (Figure 5C). In contrast, the phosphorylation site DNM1L\({}^{\text{P656/total}}\) expression level was considerably lower in the DNM1L OS group compared to the IgG OS (Figure 5D). Decreased DNM1L\({}^{\text{P656}}\) level might indicate increased mitochondrial fission [44]. Since the two phosphorylation sites synergistically regulate DNM1L activity, it is not sufficient to consider the expression level separately but to compare the ratios of both phosphorylation sites in combination (Figure 5E, DNM1L\({}^{\text{P635/P656}}\)). This ratio was moderately but significantly increased in the DNM1L OS group compared with IgG OS, indicating increased mitochondrial fission. However, the DNM1L function is determined by several pathways, not only by PTMs. Therefore, it was necessary to analyze the changes in protein expressions in retinal tissues to discuss possible molecular mechanisms and effects in retinal cells treated with anti-DNM1L. Quantitative mass spectrometric (MS) analysis and subsequent STRING pathway analysis revealed that some significantly altered proteins could be subdivided into the three protein clusters vesicle trafficking, cytoskeleton-associated, and mitochondrion-associatedsignaling. The latter is of particular interest due to the mitochondrial association of DNM1L and includes the proteins HSPA9, ANT2, and GLUD1 (Table 1). In our study, heat shock protein A9 (HSPA9) was significantly increased in the DNM1L OS group compared with the IgG OS.