Coenzyme A Levels Influence Phospo-Twinstar Levels Figure 5. HoPan induces accumulation of phospho-cofilin and affects neurite formation in neuronal differentiation in vitro. (A) Undifferentiated and retinoic-acid (RA) differentiated SHSY-5Y cells were lysed and levels of total and phosphorylated-cofilin were assayed using Western blot analysis with specific antibodies. Representative blots are shown. GAPDH was used as an additional loading control. Levels of p-cofilin were quantified from 2 independent experiments. The p-cofilin/cofilin ratio was set as 1 in mock treated cells. (B) RA-differentiated SHSY-5Y cells were additionally treated with HoPan or HoPan and pantethine. Levels of p-cofilin were assayed and quantified as in A. (C) Control SHSY-5Y cells were differentiated with RA, plated on poly-lysine and analyzed after 48 hours for their morphology (using rhodamin-phalloidin marking F-actin, red) and differentiation status (using b-III-Tubulin staining, green). In RA-treated cultures undifferentiated and differentiated cells are visible. Undifferentiated cells showed very short filopodia and did not express b-III-Tubulin (indicated as arrowheads). 48 hours after seeding many differentiated cells showed an elongated shape, neurite growth and high expression of b-III-Tubulin (indicated as arrows). (D–F) Control (D), HoPan (E) or HoPan plus pantethine (F) treated cells were differentiated with RA and plated on poly-lysine. 48 hours after seeding cells were fixed and stained for b-III-tubulin, representative parts of cell morphology and b-III-Tubulin staining of the different conditions are shown. The percentage of neurite-bearing cells was calculated for each condition. Scale bars represent 25 mm (C–F). (G) Quantification of cells showing b-III-Tubulin positive neurites is presented. doi:10.1371/journal.pone.0043145.g005 signaling pathways to brain disorders, especially to mental retardation [47,48]. Moreover cofilin activity itself has recently been reported to regulate neuronal migration [49], spine morphology [50] and synaptic plasticity [51] in mice. In humans, abnormal expression of LIM-kinase1 is associated with a mental disorder with profound cognition deficits [52]. Together these and our studies underscore the physiological importance of actin regulation and suggest that mutations that disrupt normal PANK activity (as in PKAN) can lead to altered actin dynamics and neuronal dysfunction. It needs to be pointed out that altered CoA metabolism does also influence other cytoskeletal components because recently we demonstrated that tubulin acetylation is strongly decreased in Drosophila and human cell models of PKAN (Siudeja et al., 2011). However, cell spreading and neurite formation (as measured here) depend mostly on the reorganization of the actin cytoskeleton, whereas microtubule polymerization is required for the elongation of already existing neurites [26,53]. Additional studies will be required to investigate whether cofilin PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43145