Erythrocyte membrane domains in neuroacanthocytosis

Neuroacanthocytosis (NA) is a rare, neurodegenerative disease with concomitant appearance of spiky red blood cells, known as acanthocytes. These red cells are important for NA diagnosis, however, nothing is known about the molecular mechanisms that generate them. Equally, there is nothing known about the mechanisms leading to NA- specific neurodegeneration. NA-associated mutations in the genes for VPS13A/chorein and Kx (Kell blood group system) have been identified. Both genes are expressed in the brain and red blood cell but the functional relevance for NA is not clear. The normal red cell membrane undergoes dramatic changes during terminal erythropoiesis due to massive autophagy. A defect during this process leads to an aberrant morphology as in acanthocytosis. Autophagy is also important for the clearance of toxic protein aggregates in neuronal cells. A respective defect eventually leads to neurodegeneration. Therefore, we hypothesise that a defect in the autophagic pathway involving VPS13A or Kx may account for both NA phenotypes in the brain and red cell. In this project we will focus on the red cell membrane as a model to study the basic NA disease mechanisms. The NA acanthocytes have a stable, star-like morphology and this implies the presence of a stable heterogeneity in or at the NA red cell membrane. This heterogeneity can be seen as large membrane domains that are probably generated by the association of membrane protein and lipid complexes together with the underlying cytoskeleton. In a first approach, we plan to analyse and compare relevant membrane markers by immunofluorescence microscopy (IFM) of NA and normal red cells. In particular, the distribution of scaffolding proteins and other components that are known to aggregate into high molecular complexes in the membrane will be studied. Concomitantly, the localization of normal and defective VPS13A and Kx will be determined. As a result of these studies, we assume to identify markers for the acanthocytic spikes. In a second step, we will take a biochemical approach to isolate the acanthocyte membrane domains by solubilization and fractionation by density gradient centrifugation. The composition of the isolated fractions will be determined by immunoblotting and mass spectrometry. In particular, we will focus on the distribution of acanthocyte spike markers and possible interactions with VPS13A and Kx. Associated proteins will be identified by immunoprecipitation and proteomic analysis. This information may already lead to a mechanistic insight. In a third topic we plan to study the fate of VPS13A and Kx in late erythropoiesis, particularly their possible involvement in autophagy. We hypothesize that VPS13A may play a role as a tethering component attached to autophago(lyso)somes. Mutated VPS13A may be non-functional and thus cause defective autophagy and consequently misshaped red cells. The Kx protein may be involved in the metabolic regulation of autophagosome formation. Therefore, we plan to investigate the autophagic pathway in normal and NA reticulocytes. This will be carried out by IFM co-localisation of autophagosomal markers, VPS13A, and Kx, over time. To study the loss of function of VPS13A and Kx in this context, we plan to knock- down their expression in late erythroid precursors and investigate autophagosome formation and maturation. This could eventually give rise to the formation of acanthocytes. In this project, there is a unique chance to study the biochemical basis of NA-associated acanthocyte formation that may be relevant also for neurodegeneration. The potential role of defective autophagy in the development of NA may eventually lead to the development of drugs correcting the genetic defect.

This subproject of the European Multidisciplinary Initiative on Neuroacanthocytosis (EMINA) was aimed at the investigation of red blood cells from patients suffering from neuroacanthocytosis (NA), a rare, hereditary, devastating neurodegenerative disease that is characterized by the presence of misshaped, thorny red cells (so-called acanthocytes) in the patients blood. It is plausible to assume that the same defective mechanisms leading to acanthocyte formation are also leading to neurodegeneration. Therefore, the red cell defect may lead us to the nerve cell defect. From a practical point of view, red cells are much easier to collect and study than nerve cells.A major advantage of this international co-operation project EMINA was the exchange of patients blood samples from European countries due to the help of our partners but also due to patients organizations, particularly the NA-Advocacy, NBIA Disorders Association, and Hoffnungsbaum e.V.. We also received blood from the USA and the Caribbean due to the personal engagement of Dr. Claudia Roos. She examined these blood samples by microscopic methods, assessed acanthocytosis, and analysed the proteins in the acanthocyte membranes and membrane domains. This rather trivial procedure turned out to be hampered by technical problems due to the low expression of NA-relevant proteins VPS13A and XK, the high background fluorescence of red cells, and the high individual blood donor variability. The biochemical data on protein composition of membrane domains showed many differences between normal red cells and acanthocytes but also between acanthocytes from different donors. Therefore it was not possible to assign one protein to acanthocyte formation. Our immunoblot analyses of VPS13A revealed a strong interaction of this protein with the plasma membrane similar to integral membrane proteins; however, we have not found an indication for extracellular portions upon proteolysis of intact red cells. It is possible that the strong interaction with the membrane is based on protein palmitoylation. The main focus of our biochemical/physiological analyses, in collaboration with Ulrich Salzer (MFPL/MUV), was put on functional studies that showed differences between acanthocytes and normal red cells. A prominent difference was found in the endovesiculation reaction when cells were treated with very low concentrations of antimalarial drugs. Apparently, the mechanism for endovesiculation is impaired in acanthocytes. This indicates differences in the homeostasis and regulation of cytoskeleton. Moreover, induction by lysophosphatidic acid (LPA) of red cell phosphatidylserine-exposure on the outer membrane leaflet, concomitant with calcium influx, showed a significant reduction in both reactions and indicates alterations in signalling pathways. Regarding our studies on terminal erythropoiesis and knockdown of the relevant proteins VPS13A and XK, we collaborated with the Austrian Red Cross Blood Centre in Linz and our associated partner, Mario Mairhofer (AKH/MUV), who is an expert in stem cell cultivation and erythroid differentiation. The VPS13A and XK knockdown studies are continued in the Mairhofer lab within the follow-up project EMINA-2.