Integrated analysis of novel signal transduction pathways in red cells from patients with neuroacanthocytosis

Neuroacanthocytosis (NA) is a group of rare genetic disorders that share similar neurological clinical manifestations and the presence of thorny red cells in the peripheral circulation, the acanthocytes. The two core NA diseases are Chorea-Acanthocytosis (ChAc) and McLeod Syndrome (MLS). Since acanthocytes are an hallmark of NA, studying the mechanisms underlying the generation of acanthocytes might shed light on the pathogenesis of NA syndromes. Here, we present a set of studies on the signaling mechanisms and structural changes in red cells from ChAc and MLS patients. In the first study, we evaluated tyrosine phosphorylation of red cells from ChAc patients by proteomics analysis. Increased Tyr-phosphorylation state of several membrane proteins including band 3, β-spectrin and adducin was found in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs band 3 Tyr-phosphorylation by Lyn was independent of the canonical Syk mediated pathway. The ChAc-associated alterations in RBC membrane-protein organization appear to be the result of increased Tyr-phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton. We propose this altered association between cytoskeleton and membrane proteins as a novel mechanism in the generation of acanthocytes in ChAc. In the second study, we combined phosphoproteomics datasets on ChAc and MLS with network topology analysis to predict signaling sub-networks involved in acanthocyte generation. We identified all the interactomic shortest paths linking the two proteins mutated in NA syndromes, respectively chorein and XK, to the differentially phosphorylated proteins in our proteomics data. Then, we refined the analysis considering only restricted clusters of highly interacting signaling proteins which can be involved in acanthocyte formation in both diseases. We identified a cluster of 14 kinases that might be related to red cell shape alterations and deserve further investigation. As preliminary study in the context of an international collaboration we analyzed red cells from Neurodegeneration with Brain Iron Accumulation (NBIA) patients and their first degree relatives. Our aim was to assess the presence of acanthocytes in these subjects and to study their structural characteristics. In the last study, we validated a new co-polymer based on acrylamide and polyvinyl alcohol bearing olefinic moieties in proteomic analysis of red cells. This new hydrogel is easy to handle and its macroporosity makes it suitable for the separation of high molecular weight proteins such as chorein.