Analysis of VPS13A function in iPS-derived erythroid cells (EMINA-2)

Chorea-Acanthocytosis (ChAc) is a rare, fatal neurodegenerative disease, which was shown to be caused by mutations in the VPS13A gene. Although the causative gene has been identified, the pathogenesis of the disease remains enigmatic. This is in part due to some features of the VPS13A protein, but also due to the small number of patients world-wide, which hindered research on this disease. A characteristic feature of ChAc patients are deformed, star-shaped erythrocytes (called acanthocytes) in peripheral blood, which often are the first indication of the disease. The VPS13A protein, which is most probably involved in protein transport to/from lysosomes, is expressed in neurons and erythrocytes. Therefore, mutations in the VPS13A gene are considered to be responsible for the neurodegenerative and acanthocytic phenotype. Primary human neurons of ChAc patients are not accessible for ex-vivo expansion and analysis, but patient- specific iPS cells were generated during the EMINA project and can now be used to advance our knowledge on ChAc. Neuronal differentiation of patient-specific iPS cells has been demonstrated. Nevertheless, generation of high numbers of neurons (e.g. for biochemical analysis) is not feasible. Therefore, we will differentiate iPS cells into erythroid cells in vitro to complement the fundamental research on neurons performed by our partners. Generation of erythroid cells from iPS cells has been accomplished, and these cells are highly proliferable, allowing the generation of high cell numbers and enabling biochemical and cell biological experiments not possible in neuronal cells. Our first project aim is to establish a protocol for the differentiation of patient-specific iPS cells with known mutations in VPS13A into mature erythrocytes in-vitro. These erythrocytes will be analysed for morphological and functional defects, using methods developed in the predecessor project EMINA (e.g. phalloidin staining and confocal microscopy, drug-induced endovesiculation). The iPS cell approach also allows us to study endosomal/lysosomal transport in immature, nucleated erythroid progenitor cells. The successful biochemical analysis of ChAc patient blood samples, which was started in the EMINA project, will be continued and extended. In addition, we will perform a fundamental analysis of VPS13A expression (mRNA and protein levels), sub- cellular localization and function during erythroid differentiation in vitro. To analyse functional properties, we will perform siRNA or shRNA-mediated VPS13A knock-down experiments. Identification of potential VPS13A binding partners will be performed by transfection of erythroid progenitor cells with tandem-affinity-purification (TAP)-tagged full-length or truncated VPS13A constructs, followed by purification of protein complexes and identification of candidate interacting proteins by mass spectroscopy. To identify signaling pathways potentially affected in ChAc, we will compare patient iPS cell derived erythroid cells (vs. healthy control iPS-derived cells) to screen for signaling pathways which are altered in ChAc erythroid cells. Proteome Profiler arrays (Phospho-Kinase array and Receptor Tyrosine Kinase array, R&D Systems) will be used for this purpose. These experiments will add to the understanding of altered cellular signaling in ChAc and aim to further define the up-stream signaling events leading to Lyn hyperactivation (and possibly to identify other down-stream pathways affected in ChAc).

In the international, collaborative research project EMINA-2, research groups with different expertise teamed up to gain insights into the extremely rare, severe neurodegenerative disease neuroacanthocytosis. Currently, treatment options are purely palliative, with clinicians trying to soften the inevitable, finally fatal syndromes. The genetic cause for neuroacanthocytosis, mutations in the VPS13A gene, are known for more than 15 years, but the pathogenesis is not really understood. A detailled knowledge of the function of VPS13A in the body, especially in the nervous system and in hematopoietic cells, is fundamental for the development of targeted therapies for this disease. At the Medical University of Vienna, we first focused on the function of VPS13A in erythrocytes, and could clarify some ambiguities about VPS13A by biochemical experiments. Our results clearly demonstrated that VPS13A is a peripheral membrane protein without transmembrane domains that, nevertheless, is strongly associated with the membrane. In the next phase, we decided to further investigate VPS13A function in an in-vitro cell culture model, where we could show that removal/reduction of VPS13A causes an increased adhesion of the cells to the extracellular matrix protein fibronectin, thereby affecting cell motility. We also found that signal transduction via the Focal Adhesion Kinase (FAK) and Extracellular Signal Related Kinase (ERK) is strongly affected. These results can bridge the observed phenotype to previous results showing that the actin cytoskeleton as well as autophagy are altered in VPS13A-deficient cells, and will finally lead to a better understanding of the disease phenotype. The adhesion phenotype was also observed in primary cells from neuroacanthocytosis patients. In the course of the international collaboration, we were able to work with patient-specific induced pluripotent stem cells (iPS) together with project partners in Dresden, and differentiate these cells towards hematopoietic cells. Finally, a short-term fellowship at Mount Sinai University in New York with Ruth Walker, one of the leading experts in the field, allowed me to analyse brain samples of neuroacanthocytosis patients by immunohistochemistry. Thereby, for the first time, we observed protein aggregates of poly-ubiquitinylated proteins in patient samples. This result could well transform the clinical consideration of neuroacanthocytosis, which is currently known as a neurodegenerative disease without protein aggregates.