Profilin2a-phosphorylation as a regulatory mechanism for actin dynamics.

Profilin is a major regulator of actin dynamics in multiple specific processes localized in different cellular compartments. This specificity is not only meditated by its binding to actin but also its interaction with phospholipids such as phosphatidylinositol (4,5)-bisphosphate (PIP2 ) at the membrane and a plethora of proteins containing poly-L-proline (PLP) stretches. These interactions are fine-tuned by posttranslational modifications such as phosphorylation. Several phospho-sites have already been identified for profilin1, the ubiquitously expressed isoform. However, little is known about the phosphorylation of profilin2a. Profilin2a is a neuronal isoform important for synapse function. Here, we identified several putative profilin2a phospho-sites in silico and tested recombinant phospho-mimetics with regard to their actin-, PLP-, and PIP2 -binding properties. Moreover, we assessed their impact on actin dynamics employing a pyrene-actin polymerization assay. Results indicate that distinct phospho-sites modulate specific profilin2a functions. We could identify a molecular switch site at serine residue 71 which completely abrogated actin binding-as well as other sites important for fine-tuning of different functions, for example, tyrosine 29 for PLP binding. Our findings suggest that differential profilin2a phosphorylation is a sensitive mechanism for regulating its neuronal functions. Moreover, the dysregulation of profilin2a phosphorylation may contribute to neurodegeneration.

ERK and ROCK functionally interact in a signaling network that is compensationally upregulated in Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA) is a motoneuron disease caused by low levels of functional survival of motoneuron protein (SMN). Molecular disease mechanisms downstream of functional SMN loss are still largely unknown. Previous studies suggested an involvement of Rho kinase (ROCK) as well as the extracellular signal-regulated kinases (ERK) pathways in the pathomechanism. Both pathways are bi-directionally linked and inhibit each other. Thus, we hypothesize that both pathways regulate SMA pathophysiology in vivo in a combined manner rather than acting separately. Here, we applied the repurposed drugs, selumetinib, an ERK inhibitor, and the ROCK inhibitor fasudil to severe SMA mice. Thereby, separately applied inhibitors as well as a combination enabled us to explore the impact of the ROCK-ERK signaling network on SMA pathophysiology. ROCK inhibition specifically ameliorated the phenotype of selumetinib-treated SMA mice demonstrating an efficient ROCK to ERK crosstalk relevant for the SMA pathophysiology. However, ERK inhibition alone aggravated the condition of SMA mice and reduced the number of motoneurons indicating a compensatory hyper-activation of ERK in motoneurons. Taken together, we identified a regulatory network acting downstream of SMN depletion and upstream of the SMA pathophysiology thus being a future treatment target in combination with SMN dependent strategies.

Profilin2 regulates actin rod assembly in neuronal cells.

Nuclear and cytoplasmic actin-cofilin rods are formed transiently under stress conditions to reduce actin filament turnover and ATP hydrolysis. The persistence of these structures has been implicated in disease pathology of several neurological disorders. Recently, the presence of actin rods has been discovered in Spinal Muscular Atrophy (SMA), a neurodegenerative disease affecting predominantly motoneurons leading to muscle weakness and atrophy. This finding underlined the importance of dysregulated actin dynamics in motoneuron loss in SMA. In this study, we characterized actin rods formed in a SMA cell culture model analyzing their composition by LC-MS-based proteomics. Besides actin and cofilin, we identified proteins involved in processes such as ubiquitination, translation or protein folding to be bound to actin rods. This suggests their sequestration to actin rods, thus impairing important cellular functions. Moreover, we showed the involvement of the cytoskeletal protein profilin2 and its upstream effectors RhoA/ROCK in actin rod assembly in SMA. These findings implicate that the formation of actin rods exerts detrimental effects on motoneuron homeostasis by affecting actin dynamics and disturbing essential cellular pathways.