The main findings of the current study are: (1) CK1δ and CK1ε are localized in neurites and growth cones of PC12 cells and regenerating RGCs, (2) CK1δ/ε activity is increased in differentiating PC12 cells and regenerating retina and (3) CK1-specific small molecule inhibitors destabilize existing growth cones and compromise neurite growth of RGCs and PC12 cells. These observations suggest that the activity of CK1δ and ε is essentially involved and necessary for neurite outgrowth and regeneration.
Cellular kinases and phosphatases are involved in complex signaling during neuronal degeneration and regeneration leading to remodeling of the cytoskeletal architecture 
. Members of the CK1 family reportedly play an important role in cytoskeletal rearrangements (reviewed in 
) by mediating hyperphosphorylation of the microtubule-associated protein tau, which is associated with Alzheimer's disease 
. Despite the role of CK1 family members in neurodegenerative disorders, an involvement of these kinases in neurite growth or axonal regeneration has not yet been reported.
Here, we first analyzed the localization of CK1δ and ε in primary, mature RGCs and PC12 cells. Both CK1 isoforms were located in the soma and were aligned in granular structures along microtubules. In addition, we found both isoforms located in the growth cones of regenerating RGCs and PC12 cells. Second we analyzed the occurrence of changes in the expression and activity of CK1δ and ε in regenerating RGCs and differentiating PC12 cells. No notable changes in the expression levels of CK1δ and ε were detected in retinas after optic nerve injury or when RGCs entered into a regenerative state after additional LI. NGF stimulated PC12 cells showed only a slight and temporary upregulation of CK1δ expression and a transient decrease in the expression of CK1ε. However, a 3-fold increase in the CK1δ/ε specific kinase activity was observed in differentiating PC12 cells compared with untreated controls. The CK1δ/ε specific kinase activity was confirmed by its reduction in the presence of small molecule inhibitors specific for CK1δ and ε. These data suggest that the increased CK1δ/ε activity is not basically regulated by the expression levels of CK1δ and ε, but rather through an alternative mechanism. Similar findings were also made in retinal tissue derived from untreated rats and animals that were subjected to ONC+LI. However, CK1δ/ε kinase activities detected in the current study were eluted at a higher NaCl concentration most likely due to changes in the phosphorylation state of CK1δ and ε proteins. Such changes in the chromatographic properties of CK1δ and ε after an isolation from various tissues and cell lines are consistent with previous reports 
. The activity of CK1δ is reportedly modulated by several cellular kinases, such as PKA, specifically phosphorylating CK1δ within its C-terminal domain 
. Coherently, we found a reduction in the activity of cellular CK1δ C-terminal targeting kinases in NGF differentiated PC12 cells, suggesting that the increased activity of CK1δ may have been mediated indirectly through altered activity of CK1δ C-terminal targeting kinases. However, additional experiments are necessary to identify this (these) cellular kinase(s) and to characterize their physiological interactions with CK1δ and their role in regulating neurite outgrowth.
Finally, in the current study we demonstrate that pharmacological inhibition of CK1δ/ε activity by two different ATP-competitive small molecule inhibitors markedly compromised neurite outgrowth and induced a destabilization of neurite growth cones of RGCs and PC12 cells. CKI-7 and IC261 effectively blocked neurite outgrowth of regenerating RGCs and PC12 cells at concentrations that did not affect the survival of these cells. Moreover, the fact that all tested CK1δ/ε specific inhibitors, namely IC261, CKI-7 and D4476, exhibited similar results on neurite growth minimizes the possibility of unspecific effects 
. Thus, the results of the pharmacological inhibition of CK1 imply that CK1δ/ε activity is necessary for neurite outgrowth of primary RGCs. This assumption was further underlined by time-lapse analyses showing that the CK1δ and ε specific inhibitors IC261 and D4476 induced a collapse and retraction of growing neurites of isolated RGCs, probably due to a destabilization of the microtubule and/or actin cable network. This hypothesis is supported by previous observations showing that CK1 is involved in regulating both microtubule and actin filament dynamics 
In order to obtain more information regarding the possible role of CK1 family members in maintaining microtuble integrity their functions in modulating the interaction of α/β-tubulin with microtubule associated proteins (MAPs) should be analyzed more in detail in the future. Additional experiments are also required to clarify the role of CK1δ and ε in regulating actin filament dynamics.
Although the current study suggests that CK1δ/ε activity is required for neurite outgrowth further studies need to be performed to test the possibility as to whether enhancing the activity of CK1δ and ε may facilitate neurite outgrowth and be useful for the development of therapeutic concepts to stimulate axonal regeneration. These experiments may become possible when specific activators either for CK1δ or ε will be available in the future.