Lucia De Franceschi & Acanthocytes
Search for critical VPS13A mutations in the Munich chorea-acanthocytosis (ChAc) patient cohort, continuation of chorein Western blot service, revitalization of ChAc database and transfer of all Munich achievements to the Rostock working group (Hermann & Peikert) by June 2023 – continuation of work started March 1, 2019
Adrian Danek and Gabriel Miltenberger-Miltenyi in Munich
With the support of the Advocacy, on March 1, 2019 we started our project at the Department of Neurology, LMU, Munich, and presented the project plan at the Trustees meeting in London on April 10, 2019 to the Advocacy.
The main objectives of the project, namely a. Revitalization of the chorein Western blot; b. Revitalization of the NA database and c. Genetic analysis of the ChAc-responsible VPS13A gene in blood samples available at LMU, were achieved in time, in spite of the intercurrent corona crisis. In these analyses we integrated the newest methods and elaborated new protocols. We sent out the written patient reports created to the physicians in charge.
We also achieved these additional points: (i) nominated as curators for the LOVD genetic database (https://databases.lovd.nl/shared/genes/VPS13A), (ii) became co-author on manuscript about phenotypic variability in chorea-acanthocytosis associated with novel VPS13A mutations, (iii) clarified the question about the diagnosis of PKAN in two siblings from the UK that had originally been labelled as ChAc, (iv) helped the elaboration of the scientific program of the 10th International meeting in Neuroacanthocytosis, Barcelona, 2021, (v) collaborated with our Rostock colleagues on lipid alterations in patients with ChAc, (vi) collaborated with the LMU Department of Nuclear Medicine about MLS patients, (vii) collaborated with colleagues from the Theoretical Medicine and Biosciences, Saarland University, in the biophysical-biochemical analyses of erythrocytes in ChAc.
Our next short-term (6-months) goals include the (a) continuation of Western blot diagnosis with comparative studies about antibodies and protocols, (b) completion of genetic diagnostics, (c) renovation and upgrading of our database with regard to European data protection regulations, (d) support the NA community (patients, physicians and caregivers), (e) completion of the LOVD mutation database and set up the ClinVar mutation database for VPS13A, (f) describe the causes of false negative Western blot findings, (g) completion of manuscript with clinical diagnostic guidelines for ChAc, (h) research on the zebrafish model of ChAc and MLS. We aim to continue these diagnostic and research studies until June 2023, when our scientific knowledge, samples, protocols, etc., because of Prof. Danek ́s retirement after March 31, 2023, will be transferred to our colleagues in Rostock.
In the first part of our project (March 2019 – August 2020) we managed to revitalize the chorein testing and carry out genetic analyses on patients with suspected ChAc. With newly elaborated versions of written reports as well as with continuous feed-back, we supported both patients and physicians in charge. We created new laboratory protocols that we can share with other labs, on request. With our multidisciplinary collaboration (Neurology-Clinical Genetics-Basic research) we managed to establish our centre as a core reference for ChAc.
The physiopathology of Neuroacanthocytosis Syndromes
Ruth Walker, John Crary, Melissa Nirenberg and Amber Tetlow in New York City
The core neuroacanthocytosis (NA) syndromes, namely chorea-acanthocytosis (ChAc) and McLeod syndrome, are genetically distinct, but characterized by remarkable similarities both clinically and neuropathologically. Neuropathologically, many other neurodegenerative disorders have been characterized by proteinaceous inclusions, which have driven research into underlying pathophysiological mechanisms, however, there is a dearth of such observations in the NA field. The primary neuropathological observations reported to date in ChAc have documented marked neuronal loss and gliosis. This gliosis appears to be the most significant finding, and appears to be more significant than, for example, that seen in Huntington’s disease1,2. This may potentially be an indicator of specific involvement of glial cells in ChAc.
Neuropathological studies have been limited by the rarity of these disorders, and also by the heterogeneity of methodologies used to study cases from different academic centers.
In this proposal we will perform systematic neuropathological studies on tissue from a series of ChAc and McLeod syndrome brains, informed by recent advances in research in the field, in addition to utilizing recent methodological developments in neuropathology. We will also perform analysis of lipid composition of these tissues; data from erythrocytes has indicated abnormal sphingolipid composition, which may be related to the abnormal membrane conformation which results in acanthocytosis, and may be related to neuronal degeneration.
The core neuroacanthocytosis (NA) syndromes, namely chorea-acanthocytosis (ChAc) and McLeod syndrome, are genetically distinct, but look very similar when brain tissues are examined down the microscope after death. The brains of people who suffered from a number of other neurodegenerative disorders, such as Parkinson’s, Alzheimer’s, and Huntington’s diseases have been found to contain clumps of protein, both inside and outside neurons. Analysis of the protein clumps has been informative in driving research into these disorders. NA syndromes are unusual in that such protein clumps have not been identified. In fact, the main observations in people with NA has been that there is loss of brain cells in specific regions, and a lot of scar tissue, known as “fibrosis”. We propose to use the latest neuropathological techniques, informed by recent research in the field, to look for other clues as to what is killing brain cells in these diseases. We will also be looking at constituents of lipids (fats) in the brain, as abnormalities of these may underlie the abnormal red cell shapes, and may also provide insights into brain cell damage.
Cardiac manifestation is evident in chorea-acanthocytosis but different from McLeod syndrome
Silvio Quick, Felix Matthias Heidrich, Max-Valentin Winkler, Anna Helene Winkler, Karim Ibrahim, Axel Linke, Uwe Speiser, Ulrich Grabmaier, Carsten Buhmann, Franz Marxreiter, Carsten Saft, Adrian Danek, Andreas Hermann, Kevin Peikert
Introduction: We aimed to study the various cardiac manifestations of the two core neuroacanthocytosis (NA) syndromes, namely chorea-acanthocytosis (ChAc) and McLeod syndrome (MLS). So far, cardiac involvement has been described as specific feature only for MLS.
Methods: We studied six patients with ChAc (mean age 44.5 years, five men, one woman) and six patients with MLS (mean age 57.1 years, all men). Cardiac evaluation included echocardiography and/or cardiac magnetic resonance imaging (cardiac MRI), 24-h ECG-recording and examination of cardiac biomarkers.
Results: Cardiac involvement of ChAc was found in four of six patients. Two patients showed mildly reduced left ventricular ejection fraction (LVEF), two other patients mild to moderate left ventricular (LV) dilatation. Neither an increase in ventricular ectopic beats nor ventricular tachycardia were evident in ChAc. Four of five MLS patients showed left ventricle dilatation and reduced left ventricular ejection fraction (LVEF). Two of these, in addition, had critical ventricular tachycardia. High sensitive troponin T was elevated in all patients, for whom data were available (n = 10). In contrast, elevation of high sensitive troponin I was found in one of six ChAc and one of two MLS patients.
Conclusion: For the first time, we reveal cardiac involvement in a cohort of six ChAc patients, while the risk to develop heart failure seems lower than in MLS. Our study confirms the malignant nature of MLS in terms of ventricular arrhythmias and progression to advanced heart failure. Herein, we define disease-specific recom- mendations for cardiac assessment in both conditions.
Acanthocyte Sedimentation Rate as a Diagnostic Biomarker for Neuroacanthocytosis Syndromes: Experimental Evidence and Physical Justification
Alexis Darras, Kevin Peikert, Thomas John, Anil Kumar Dasanna, Antonia Rabe, François Yaya, Greta Simionato, Semen Buvalyy, Jürgen Geisel, Andreas Hermann, Christian Wagner and Lars Kaestner
The binding of the APT1 domains to phosphoinositides is regulated by metal ions in vitro
Damian Kolakowski, Joanna Kamińska, Teresa Zoladek.
Biochimica et Biophysica Acta (BBA)-Biomembranes, 183349, 2020
Decreased Na+/K+ ATPase expression and Depolarized cell Membrane in neutrons Differentiated from Chorea-Acanthocytosis Patients
Zohreh Hosseinzadeh, Stefan Hauser, Yogesh Singh, Lisa Pelzl, Stefanie Schuster, Yamini Sharma, Philip Hoeflinger, Nefeli Zacharopoulou, Christos Stournaras, Daniel L. Rathbun, Eberhart Zrenner, Ludger Schoels, Florian Lang
Scientific Reports 10 (1), 1-10, 2020
Identification of two compound heterozygous VPS13Alarge deletions in chorea‐acanthocytosis only by protein and quantitative DNA analysis
Derek Spieler, Antonio Velayos‐Baeza, Alžbeta Mühlbäck, Florian Castrop, Christian Maegerlein
Julia Slotta‐Huspenina, Benedikt Bader, Bernhard Haslinger, Adrian Danek
Wiley Online Library 14 February 2020
Yeast-model-based study identified myosin-and calcium-dependent calmodulin signalling as a potential target for drug intervention in chorea-acanthocytosis
Piotr Soczewka, Damian Kolakowski, Iwona Smaczynska- de Rooij, Weronika Rzepnikowska, Kathryn R Ayscough, Joanna Kamińska, Teresa Zoladek.
Disease models and mechanisms 12 (1), 2019
Huntingdon’s disease-like disorders in Latin America and the Caribbean
Ruth H Walker, Emilia M Gatto, M Leonor Bustamante, Oscar Bernal-Pacheco, Francisco Cardoso, Raphael M Castilhos, Pedro Chana-Cuevas, Mario Cornejo-Olivias, Ingrid Estrada-Bellmann, Laura B Jardim, Ricardo Lopez-Castellanos, Ricardo Lopez-Contreras, Debora P Maia, Pilar Mazzetti, Marcelo Miranda, Mayela Rodriguez-Violante, Helio Teive, Vitor Tumas.
Parkinsonism and Related Disorders 53, 10-20, 2018.
Pathoarchitectonics of the cerebral cortex in chorea‐acanthocytosis and Huntington's disease
J. Liu, H. Heinsen, L. T. Grinberg, E. Alho, E. Amaro Jr, C. A. Pasqualucci, U. Rüb, K. Seidel, W. den Dunnen, T. Arzberger, C. Schmitz, M. C. Kiessling, B. Bader, A. Danek
Wiley Online Library 2 May 2018
Molecular basis and clinical overview of McLeod syndrome compared with other Neuroacanthocytosis syndromes: a review
Eileen Roulis, Catherine Hyland, Robert Flower, Christoph Gassner, Hans H Jung, Beat M Frey
Jama Neurology 75 (12), 1554-1562, 2018
Current state of knowledge in Chorea-Acanthocytosis as core Neuroacanthocytosis syndrome
Kevin Peikert, Adrian Danek, Andreas Hermann
European Journal of medical genetics 61 (11), 699-705, 2018
Amino Acid substitution equivalent to human chorea-acanthocytosis l2771R in yeast VPS13 protein affects its binding to phosphatidylinositol 3- phosphate.
Weronika Rzepnikowska, Krzysztof Flis, Joanna Kaminska, Marcin Grynberg, Agnieszka Urbanek, Kathryn R Ayscough, Teresa Zoladek.
Human Molecular Genetics 26 (8), 1497-1510, 2017
Neuronal dysfunction in iPSC-derived medium spiny neurons from chorea-acanthocytosis patients in reversed by Src kinase inhibition and F-actin stabilisation.
Nancy Stanslowsky, Peter Reinhardt, Hannes Glass, Norman Kalmbach, Maximilian Naujock, Niko Hensel, Verena Lübben, Arun Pal, Anna Venneri, Francesca Lupo, Lucia De Franceschi, Peter Claus, Jared Sterneckert, Alexander Storch, Andreas Hermann, Florian Wagner
Journal of Neuroscience 36 (47), 12027-12043, 2016
VSP13Aand Autophagy, possible connections and preclinical studies
Ricardo Escalante for Sandra Munoz, Biomedical Research Institute Alberto Sols, Madrid
Application of new Antibodies on chorein research
Antonio Veleyos Baeza, Welcome Trust Centre for Human Genetics, Oxford
I. FUNCTION OF VPS13A (CHOREIN), THE PROTEIN ABSENT IN CHOREA-ACANTHOCYTOSIS PATIENTS (ChAc)
A.) Vps13A regulation of phosphatidylinositolphosphate pools in mammalian cells
Aaron Neiman, Stony Brook University, New York
The genetic disease chorea acanthocytosis is caused by mutations in the gene Vps13A in humans. Vps13A encodes an evolutionary conserved protein of unknown molecular function. We have found that during the development of spores in the baker's yeast, Saccharomyces cerevisiae, the yeast homolog of Vps13A regulates the growth of intracellular membranes by controlling the levels of specific membrane components termed phosphatidylinositol phosphates. We will examine if the mammalian protein similarly regulates phosphatidylinositol phosphates. These membrane components are involved in a variety of cellular functions. Alteration of these components in cells lacking VpsS13A could account for the symptoms of chorea acanthocytosis and suggest avenues for treatment of the disease.
B.) Study of the basic features and the role of Vps13A in human cell line models.
Antonio Velayos Baeza, Wellcome Centre for Human Genetics, University of Oxford
The main goal of this research project is to obtain a good characterisation of the basic features of Vps13A. Analysis will include the effects that some particular mutations found in ChAc patients, which are predicted to cause only small changes in Vps13A, have in the localisation, biochemical features and/or stability of this protein, and will investigate the effects of absence/knockdown of endogenous Vps13A at the sub-cellular level. The work required for this research plan will continue previous work over ten years by this group so they already have a number of tools and data available relative to Vps13A characterization which would be valuable assets for the success of this project. This work complements the research of Aaron Neiman who approaches the problem from strong experience in yeast. Antonio and Aaron will share their insights in collaboration. These studies may reveal why the absence of VPS13A causes death of neurons in the brain and provide a step towards finding a way to replace this function as a possible therapy for ChAc.
C.) Progress on Grant from the Advocacy, Purification and biochemical function of yeast Vps13 protein
Robert S. Fuller and Mithu De, Department of Biological Chemistry,U. of Michigan Medical School, Ann Arbor, MI, USA
This grant supports studies by postdoctoral fellow Mithu De on the basic biochemistry of Vps13 protein (Vps13p), the yeast homolog of the ChAc disease protein, Vps13A. Loss of function of the yeast Vps13 gene results in defects in vacuolar biogenesis, localization of trans- Golgi network (TGN) enzymes and spore membrane formation. Vesicular transport between the TGN and the late endosome (in yeast termed the PVC for "prevacuolar compartment") is required for both vacuolar biogenesis and TGN enzyme localization. We have reconstituted this vesicular transport step in a cell-free system using yeast extracts and have discovered that extracts from vps13 mutant strains are defective in this trafficking step. We could complement this defect in vitro by adding cytosol that contains wild-type (WT) Vps13p. We also confirmed that the small calmodulin-like calcium-binding protein Cdc31p interacts with Vps13p.
The two aims of the grant are (i) to purify and characterize an active form of Vps13p and (ii) to determine the role of Cdc31p in Vps13p function. Significant progress has been made on both aims. First, we have used the cell-free TGN-PVC transport reaction as an assay to follow purification of a biochemically active form of Vps13p. Scaling up this purification will permit us to identify novel associated proteins and to assess the mechanism by which this peripheral membrane protein associates with membranes. Second, we have shown that cdc31 mutant extracts are also defective for TGN-PVC transport and that this defect can be complemented by adding purified Vps13p. This result implies that Cdc31p is required for TGN-PVC transport and that the form of Vps13p that is active in TGN-PVC transport is a Vps13-Cdc31 complex. Currently we are using deletion analysis to map sequences in Vps13p required for binding to Cdc31 and for interacting with late endosomal/PVC membranes.
Report from Robert S. Fuller and Mithu De, Department of Biological Chemistry,U. of Michigan Medical School, Ann Arbor, MI, USA
Towards an understanding of the biochemical and cellular functions of Vps13 proteins.
The neurodegenerative disease Chorea Acanthocytosis is caused by mutations that inactivate the human VPS13A gene. We know that loss of the function of the VPS13A gene product results in the eventual death of striatal neurons of the basal ganglia and defects in red blood cells (acanthocytosis). A straightforward hypothesis is that VPS13A is required for a cellular process that is critical both for neuronal survival and for normal red blood cell structure. We do not know what that process is, but identifying it essential both to understanding why the neurons die and to devising possible therapeutic strategies. The approach we are taking is to study the function of an evolutionarily related gene in a model microbe, baker's yeast (Saccharomyces cerevisiae).
The name of the VPS13A gene comes from a gene in baker's yeast, VPS13, which was discovered based on its role in the selective transport of proteins between two membrane-enclosed organelles in the yeast cell termed the trans Golgi network (TGN) and the late endosome (LE). This TGN-LE pathway is essential for the maintenance of the yeast lysosome or vacuole, another membrane enclosed organelle. The lysosome acts as a kind of cellular garbage disposal, digesting cellular components such as proteins and lipids. The TGN-LE pathway is also needed to maintain the function of yet another membrane-enclosed organelle, the Golgi complex, which is required for the correctly modifying proteins that are secreted or released from cells. Mutations in the yeast VPS13 gene block the TGN-LE pathway, interfering with normal lysosome and Golgi complex function. Our laboratory originally isolated the yeast VPS13 gene in 1997 and identified the product of the gene, Vps13p, a novel and unusually large protein. We have now developed a method for reconstituting in the test tube protein trafficking between the TGN and LE using extracts of yeast cells. This assay has allowed us to measure the biochemical activity of the Vps13 protein, to show that it functions directly in TGN-to-LE trafficking and to purify the active form of the protein. Analysis of the purified protein and other proteins that associate with the active protein will give us critical information about how the Vps13 family of proteins function in cells and what molecules they communicate with. This information will allow us to formulate testable hypotheses about how VPS13A functions in human cells.
II. STUDY OF ChAc FROM iPS CELLS
A.) Invitro modelling of chorea-acanthocytosis: patient fibroblasts and their reprogrammed derivatives as human models of ChAc
Alexander Storch, Dresden Technical University, Dresden, Germany
The overall aim of this ongoing project is to establish an in vitro model of ChAc using nerve and blood cells from reprogrammed fibroblasts from patients suffering from ChAc. Furthermore, we investigated patient erythrocytes. We could identify severe cytoskeleton disturbances within erythrocytes from ChAc patients. We found decreased phosphoinostide-3-kinase (P13K)-p85-subunit phosphorylation, ras-related C3 botulinum toxin substrate 1 (Rac1) activity, and p21 protein-activated kinase 1 (PAK 1) phosphorylation leading to depolymerized cortical actin in erythrocytes from patients with ChAc and K562-erythrocytoic cells following Vps13A silencing. Moreover, Vps13A silencing and PAK1 inhibition promoted apoptosis by forming mitochondrial pores followed by mitochondrial depolarization, DNA fragmentation, and phosphatidy1serine exposure at the cell surface, all hallmarks of apoptosis.(Foller, Hermann et, FASEBJ 2012)
We succeeded to derive iPS cell lines from two patients and two healthy age- and sex- matched controls. These cell lines underwent all quality control steps needed like differentiation capacity, retroviral gene silencing, teratoma formation, micro array analysis and caryotyping. Respective clones were used for further analysis , in close collaboration with the Max Planck Institute for Molecular Biomedicine Muenster.
B.) Functional analyses of ion channels in chorea-acanthocytosis
Florian Wegner, Hannover Medical School, Germany; Alexander Storch, Andreas Hermann, Dresden Technical University
The aim of this research project is to gain insight into the functional pathomechanisms of ChAc, an autosomal-recessive neurodegenerative disease of young adults manifesting in a hyperkinetic movement disorder and other severe neurological symptoms as well as in misshaped red blood cells. This disease is caused by mutations in the Vps13A gene leading to a loss of the largely unknown function of the protein Vps13A. Recently, we established a human in vitro model of ChAc using reprogrammed skin fibroblasts from ChAc patients. The genetic transformation of easily accessible patient cells allowed the generation of disease-specific induced pluripotent stem cells (iPSC) that behave similarly to embryonic stem cells without raising ethical concerns. iPSC-derived neurons are indeed believed to be the best human cell model for neurological diseases.
In this project, the ChAc patient-derived iPSC will be differentiated into various types of neurons to study their ion channel function and synaptic activity by electrophysiological and calcium-imaging methods. Particularly, the analysis of synaptic activity in disease-specific neurons is of great interest because we recently identified severe actin-related cell membrane disturbances in erythrocytes from ChAc patients. This impairment of the cell membrane function may result in pathological synaptic currents due to the presynaptic role of the ChAc actin in neurons. We will compare the functional data of disease-specific neurons with neuronal cells derived from healthy controls in order to shed light on the functional pathophysiology and identify a potential therapeutic target to treat ChAc.
Report from Florian Wegner, Hannover Medical School, Germany 4.14
Functional analyses of ion channels in Chorea-Acanthocytosis (ChAc) patient-derived induced pluripotent stem cells and differentiated neurons in vitro.
We are trying to understand the mechanism underlying neuronal degeneration in ChAc to develop neuroprotective treatment strategies. We established a model system of ChAc using the induced pluripotent stem cell (iPSC)-technology. iPSCs are immortalized, self renewing cells which were generated from skin cells of two ChAc patients and two healthy controls by genetic modification. Those iPSCs were differentiated into medium spiny neurons predominantly affected in ChAc. By comparing the neuronal properties of cells from ChAc patients to those of healthy controls altered ion currents in ChAc neurons were recorded, which point towards a pathological excitability that is believed to account for neuronal death in ChAc. We tried to ‘rescue’ the observed phenotype by treating the cells with the sodium channel blocking drug riluzole, which is already approved for treatment of other neurological diseases like amyotrophic lateral sclerosis. Alternatively, we followed the evidence obtained from erythrocytes of ChAc patients suggesting a causal relation of the observed excitability in ChAc neurons with a destabilized actin-cytoskeleton. Therefore, we investigated the effects of the actin stabilizer phallacidin as an actin-stabilizing agent Another starting point arose from the observation of elevated Lyn kinase activity and increased protein phosphorylation in ChAc erythrocytes. Assuming exalted protein phosphorylation also in ChAc neurons, we explored the impact of the kinase inhibitor PP2 on our cells. Future studies will be conducted to confirm our preliminary results by using more iPSC lines and in order to get a more detailed insight into the pathomechanism underlying ChAc.
III. FUNCTION OF XK PROTEIN IN McLeod SYNDROME
The role of XK protein in Erythrocyte ion transport function
Alicia Rivera PhD at Children’s Hospital, Harvard Medical School, Boston, USA
This study will test the molecular mechanisms that are important in the development of acanthocytic red cells. We are currently seeking patients who have been diagnosed with lack of Kx antigen, McLeod syndrome. No medication for neurological, or other disorders such as benzodiazepines, anticonvulsants, anti-hypertensive or other cardiac medications, and antidepressants. Family/Patients: If you have been told by your doctor that you have McLeod syndrome, or that you lack the Kx antigen in your red cells, you may qualify for this research study. It is important that you are not taking any medication as this could interfere with the studies of red cells. We are only approved to recruit adults over the age of 21 years. If you qualify, participation would involve 2 visits to your doctor to give a medical and family history and to have blood samples taken. There are no medications involved. As part of the study you will be asked to contact close family members to see if they would also like to participate. You will not receive any personal health benefits as a result of your participation in this research study. However, the results will allow us to better comprehend McLeod syndrome and thus benefit patients in the future.
There are no medications involved. Researchers will isolate DNA from one blood sample, so that they can confirm the lack of Kx antigen. The study will assess cation transport across intact red blood cells and estimate how this critical red cell function is affected by the absence of XK protein.
If you would like to receive additional information about participating in this important Institutional Review Board approved research study, please contact Dr. Alicia Rivera, Principal Investigator, or Dr. Ruth Walker, patient recruiter by email: firstname.lastname@example.org or email@example.com . (The family of Mark Willard supported by the Advocacy made this grant directly.)
Alicia Rivera, MS, PhD, Boston Children’s Hospital / Harvard Medical School, Boston, USA November 2013
The McLeod syndrome is a rare genetic disease caused by an error in the XK gene (XK) in the X-Chromosome. The specific goals of this project are to identify and characterize the physiological and functional role of XK proteins in erythrocytes. We have recently reported (Blood Cell, Molecules and Diseases, Rivera et al, 2012) strong evidence of a previously un-described alteration in erythrocyte cellular magnesium and potassium ion homeostasis in cells from Xk knockout mice when compared to wild-type mice. We have now significantly expanded on these observations in rodents. Our results indicate that red cells from a young patient with XK mutation but no MLS presentation show an 18% increase in intracellular K+ (245.3 to 288.5 mmol/Kg Hb) and Mg2+ (5.6 to 7.5 mmol/Kg Hb) that was associated with lower total calcium levels (1.1 to 0.3 mmol/Kg Hb) when compared to erythrocytes from otherwise healthy subject as determined by atomic absorption spectrophotometry. These results suggest the existence of alterations in cation transporters in the cells from this patient. Indeed and consistent with this hypothesis, a more detailed investigation revealed that Na+ independent Mg2+ permeability in these red cells was likewise altered (2.0 to 0.6 mmol/1013 cell x h). In addition, we observed that the Na/Mg exchanger was significantly altered in cells from this young patient when compared to the control. Furthermore, we also evaluated K+ transport mediated by the Gardos channel (Ca2+ stimulated K flux) in the erythrocytes from this patient and observed significantly increased activity when compared to cells from healthy subject (18 to 26.8 mmol/1013 cell x h). These alterations in the Na/Mg exchanger and the Gardos channel are consistent with what we observed in the mice that lacked the Xk gene. Na+ transport was also evaluated but no significant differences were found in the Na pump, Na/K/2Cl cotransport or Na/H exchanger activity. Small changes, albeit significant, in mean cellular volume or red cell distribution width were observed in this patient. However, more patient samples should be studied to confirm these observations. These results provide two important contributions to the field, 1) they show strong evidence suggesting that Xk deletion leads to changes in the erythrocyte cation homeostasis and 2) they strongly implicated Xk protein as a novel transporter and regulator of cellular magnesium and potassium ions; alterations that might play critical roles in the development of acanthocytic red cells in patients MLS. Thus our results are very promising and bring novel insight on potential mechanisms that may in part explain the development of acanthocytic erythrocytes in patients with MLS. We thank to Prof. Dr. Hans H. Jung from University Hospital in Zürich for his kind collaboration in providing us with the first sample. Another potential young MLS candidate from the Zürich group has been located and we are waiting for patient availability. One of these patients who is categorized as asymptomatic McLeod has a E327K missense mutation in XK gene product will be included in this study. We found two additional asymptomatic McLeod brothers who have this mutation in NY area. We would like to include these McLeods patients in this study. Thus, these asymptomatic subjects are very valuable to compare the results with McLeod with MLS. Including these McLeods in the study may give us additional valuable information in finding the physiological function of XK (and Kell). We are currently seeking patients who have been diagnosed with absence of Kx antigen, McLeod syndrome, that are not on any medication for neurological or other disorders and are not taking benzodiazepines, anticonvulsants, anti-hypertensive or other cardiac medications or antidepressants. Consequently we would like to recruit young McLeod individuals who have not developed symptoms.
IV. GENETIC STUDIES AND REGISTRY DEVELOPMENT
Genetic studies of five ChAc patients to determine if there are other genetic anomalies that may interact with the absence of VPS13A to create the symptoms of ChAc
Dr. Gabriel Miltenberger-Mityeni, University of Lisbon, Lisbon, Portugal
The research will correlate the genetic findings with patients’ clinical symptoms of NA diseases as they are recorded in their pseudonymized clinical information.
V. ACANTHOCYTE MEMBRANE STUDIES
Computational identification of phospho-tyrosine sub-networks related to acanthocyte generation in neuroacanthocytosis PLoS One. 2012;7(2):e31015. Epub 2012 Feb 15
De Franceschi L, Scardoni G, Tomelleri C, Danek A, Walker RH, Jung HH, Bader B, Mazzucco S, Dotti MT, Siciliano A, Pantaleo A, Laudanna C.
Erythrocyte membrane changes of chorea-acanthocytosis are the result of altered Lyn kinase activity Blood. 2011 Nov 17;118(20):5652-63. Epub 2011 Sep 27
De Franceschi L, Tomelleri C, Matte A, Brunati AM, Bovee-Geurts PH, Bertoldi M, Lasonder E, Tibaldi E, Danek A, Walker RH, Jung HH, Bader B, Siciliano A, Ferru E, Mohandas N, Bosman GJ. Department of Medicine, University of Verona, Piazzale Lo Scuro 10, Verona, Italy. firstname.lastname@example.org
European Multidisciplinary Initiative on Neuroacanthocytosis
The rare conditions collectively labelled as neuroacanthocytosis (NA) seriously disable young adults and place a heavy burden on them and their families. NA syndromes affect about 1 in 3 million individuals and present a variety of emotional, cognitive and movement disorders. They share considerable similarities with Huntington´s disease (HD). As in HD, the basal ganglia are preferentially affected by neurodegeneration but in contrast to HD a key to the understanding of underlying mechanisms may be found in easily accessible peripheral cells.
It is characteristic for NA that deformed erythrocytes with thorny protrusions are found in the patients´ blood. Although not detectable in every single case, these cells are the origin of the term neuroacanthocytosis, denoting the association of neurological findings and acanthocytes (“akantha”: Greek for thorn).
NA syndromes may be divided into those with lipid abnormalities and peripheral nervous system involvement (such as abetalipoproteinemia) and into the “core” NA syndromes with central nervous system involvement, including basal ganglia degeneration. Currently, the genetic basis is known for four of these conditions: McLeod syndrome (MLS, X-linked), chorea-acanthocytosis (ChAc, autosomal recessive), Huntington´s disease-like 2 (HDL2, autosomal dominant) and pantothenate kinase associated neurodegeneration (PKAN, autosomal recessive). Responsible genes are the Kell protein associated XK in MLS, VPS13A (vacuolar protein sorting 13 A, chorein) in ChAc, JPH3 (junctophilin 3) in HDL2 and PANK2 (pantothenate kinase 2) in PKAN. The recognition of PKAN as part of the NA spectrum creates a link to the NBIA group of syndromes (“neurodegeneration with brain iron accumulation”). NBIA syndromes are characterized by high brain iron with typical magnetic resonance imaging findings and the presence of axonal spheroids on histology. NBIA syndromes include some genetically still undefined conditions: PLA2G mutations were most recently added to the list of genes involved. At least 8% of PKAN patients show RBC acanthocytosis yet for other NBIA syndromes such analyses are currently not available.
European Multidisciplinary Initiative on Neuroacanthocytosis (EMINA)
Little is known on the disease progression milestones and no treatment or cure of the debilitating disorders are available. Work with an international database should allow the development of scales for systematic treatment studies and the analysis of experience gained with procedures such as deep-brain stimulation (DBS) that is increasingly considered in the NA and NBIA syndromes.
Besides the actual mechanisms in neurodegeneration, the formation of the essentially unknown basis of the acanthocytic shape change is interesting to basic scientists. This implies alterations of membrane components of the outer and inner leaflet as compared to the disk shape of a normal RBC, the discocyte. These alterations imply a relative dilation of the outer membrane leaflet or a compression of the inner leaflet or a combination of both, outer leaflet dilatation and inner leaflet compression. Acanthocytic cells in NA blood appear to persist for longer. Thus, one may assume the existence of stable domains within these membranes, particularly in the “thorn” regions.
One interesting aspect concerning neurons as well as erythrocytes is the role of vesiculation and autophagy in the NA and NBIA syndromes. Chorein, the VPS13A protein, appears to be involved in such processes while terminal erythropoiesis and maintenance of (neural) cell viability are both connected to autophagy. A defect in the autophagic pathway could therefore account for both neuronal and RBC dysfunction and may be studied in vitro using patient RBC membranes. Post mortem analyses of nervous tissue in the NA and NBIA syndromes on a systematic basis, using case series, are similarly needed and require appropriate collection and preservation of tissue. Nervous tissue from ChAc cases is available in the Munich brain bank and examined neuropathologically.
The development of animal models for the various disease states has become possible, yet in comparison to HD the pace has been very slow. The few existing mouse models (for MLS, ChAc, HDL2 and PKAN, lacking variety in terms of gene mutations) have not yet been exhaustively analysed and the situation appears similar for C. elegans (MLS) and Tetrahymena thermophila (ChAc). For PKAN a drosophila model has recently become available. Development of animal models for NA and NBIA syndromes clearly is a major goal in EMINA.
LMU - Ludwig-Maximilians-University, Neurologische Klinik und Poliklinik, Munich, Germany
MUV - Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria
RUN - Radboud University Medical Center Nijmegen, Biochemistry, Nijmegen, The Netherlands
UMCG - University Medical Center Groningen, Groningen, Cell Biology, The Netherlands
CHUB - Centre Hospitalier Universitaire de Bordeaux, Service de Neurologie, Bordeaux, France
ITF - Istanbul Faculty of Medicine, Department of Neurology, Istanbul, Turkey
Diagnostic testing for ChAc is available at the University of Munich and is based on a Western blot of red blood cell (RBC) membranes. Instructions [PDF, 0,8 MB].
A dedicated databank was formed for this purpose through collaboration with the European Huntington´s Disease Network (Euro-HD) and is accessible through the internet. [...more]
EMINA aims and subprojects:
Set up NA reference centre located in Munich (Partners involved: LMU, CHUB, ITF).
Set up a diagnostic centre for ChAc located in Istanbul (Partners involved: LMU, ITF).
Analyse NA red cell membranes composition (Partners involved: MUV, RUN).
Analyse NA red cell proteoms and study localisation of NA relevant proteins in control cells and knock down effect on control cells (Partners involved: MUV, RUN).
Elucidate the mechanism of vesicle formation in erythrocytes and acanthocytes of patients with various forms of NA (Partners involved: RUN).
Develop Drosophila models for other NA syndromes in addition to the existing Drosophila PKAN model to identify underlying common mechanism of cell degeneration (Partners involved: UMCG).
Create diagnostic guidelines and taxonomy for NA syndromes including collection of NA patients (Partners involved: LMU, CHUB, ITF)