EMINA working group: Diagnostics, epidemiology, neuropathology and patients' database
Aims of the project - Hereditary neuroacanthocytosis (NA) syndromes play an important role within the differential diagnosis of patients with chorea and cryptic epilepsy. The major sub-syndromes in NA derive from mutations in various genes and to the subsequent impairment in protein function. Although major advances in genetics and molecular biology made in the recent years, the molecular functions leading into the pathological process in NA syndromes are still unknown. Though hints of protein function exist, it is mandatory to reveal the pathological process of neurodegeneration in general and the connection between malformed erythrocytes and neurodegeneration in particular in order to design new and effective therapeutic options. To carry out efficient investigations, it is crucial to establish a reliable diagnostic and a sustainable tool to monitor clinical evaluation and therapeutic impact of NA patients and to keep track on their world wide localization. This will also help to gather tissue samples of NA patients in order to support studies of neurodegeneration and general pathological changes. Despite of the rareness of NA syndromes, evaluation of their characteristics may contribute to a general understanding of different basal ganglia degeneration disorders.
Workplan - Our general goals are keep up the service of free diagnostics of chorea-acanthocytosis offered to international physicians, to administer the neuroacanthocytosis database, to collect tissue samples of patients and to support patients, physicians and basic scientists in neuroacanthocytosis issues. This includes submission of grant proposals and coordination of joint publications. Althought our project mainly focuses on chorea-acanthocytosis all other inquiries to NA syndromes are welcome and can be redirected. More specific, we are using chorea-acanthocytosis tissue material to investigate functional aspects of chorein.
EMINA working group: Clinical evaluation of NA patients
Aims of the project - NA patients will be evaluated and clinically analysed in order to develop general rating scales to pay tribute to different disease stages. The group of Prof. Tison does have extensive experience in the development of clinical rating scales and will identify milestones during the course of the different sub-syndromes in NA. Patients will not only be evaluated clinically but also according to the effectivity and efficacy of various therapeutic strategies.
Workplan - The phases of this project are as follows:
Designing severity rating scales and disease progression milestones in NA
Assessment of interventional therapies in NA
François Tison () Centre Hospitalier Universitaire de Bordeaux Service de Neurologie Pessac, France France Project Homepage: www.chu-bordeaux.fr
EMINA working group: Recruitment and molecular evaluation of NA patients in Turkey
Aims of the project - We will continuously collect blood samples of patients that are highly suspected of suffering from an NA syndrome due to the clinical phenotype or show at least an elevated level of acanthocytes in peripheral blood. Due to the large outreach of the clinic, a at least 40 NA patients of all kinds should be reached within 3 years of the project duration.
Workplan - The phases of this project are as follows:
Systematic evaluation of acanthocytes prevalence in high-interest-patients
Systematic evaluation of chorein levels in identified acanthocytosis patients
Further recruitment of NA patients
Taxonomic classification of acanthocytosis syndromes
Zuhal Yapici () Istanbul Faculty of Medicine Department of Neurology Istanbul, Turkey Project Homepage: www.itfnoroloji.org
EMINA working group: Erythrocyte membrane domains in neuroacanthocytosis
Aims of the project - The acanthocyte shape, as compared to the normal discocyte, implies alterations ofmembrane components of the outer and inner leaflet that lead to a relative dilation of the outer leaflet and/or compression of the inner leaflet. Because of the stability of the NA-based acanthocytic shape one can assume the existence of stable domains within the membrane, particularly in the spike regions.
Workplan - The phases of this project are as follows:
Microscopic analysis of NA red cell membrane domains
Biochemical analysis of NA red cell membrane domains
Analysis of autophagy during terminal erythropoiesis
Rainer Prohaska () Medical University of Vienna Vienna Max F. Perutz Laboratories Austria Project Homepage: www.meduniwien.ac.at/Neurologie
EMINA working group: Generation and validation of Drosophila models for NA
Aims of the project - Neuroacanthocytosis (NA) is linked to mutations in the following genes: VPS13A (Choreaacanthocytosis); XK (McLeod syndrome); JPH3 (HDL2) and PANK2 (PKAN). The model organism Drosophila melanogaster with its powerful genetics offers opportunities to create and investigate disease models for NA. A Drosophila disease model for PKAN has already been established and validated by us and shows several characteristics shared by PKAN patients (Bosveld et al., 2008). Additional Drosophila NA models will be generated in order to identify and understand shared underlying mechanisms leading to NA. Recently the laboratory of Giel Bosman (Bosman and De Franceschi, 2008) found indications for altered vesicle formation in erythrocytes of NA patients as a possible underlying mechanism for acanthocytosis. Neuronal tissue of patients is limited and especially the Drosophila models will be useful to investigate the possible role of abnormal vesicle behavior in neuronal cells (reviewed in Levitan, 2008).
Workplan - The phases of this project are as follows:
Development of a Drosophila model for ChAc and for HDL-2
Investigate whether altered vesicle formation is present in Drosophila NA models
Ody Sibon (
) University Medical Center Groningen (UMCG) Cell Biology/Radiation and Stress Cell Biology Groningen, The Netherlands The Netherlands Project Homepage:www.umcg.nl/NL/Zorg/Pages/Default.aspx
EMINA working group: A proteomic inventory of the erythrocytes of patients with neuroacanthocytosis I
Aims of the project - The major aim of this project is to generate a comparative
proteomic inventory of the membrane fractions of erythrocytes from NA patients
with chorea-acanthocytosis, McLeod syndrome, and Huntington disease-like 2.
Workplan - Our main approach consists of a proteomic analysis of
various membrane fractions, in combination with differential extraction in order
to separate integral membrane proteins from the cytoskeleton and from associated
soluble proteins. This approach has recently been proven to be highly efficient
and informative in a comparative proteomic analysis of erythrocytes and vesicles
of various ages and storage periods.
Dr. Giel J.C.G.M. Bosman,
Ph.D. (
.bosman@ncmls.ru.nl) Department of Biochemistry (286) University of Nijmegen Medical Centre and Nijmegen Centre for Molecular Life Sciences Nijmegen, The Netherlands Project Homepage: www.ncmls.nl
Associated working group: A proteomic inventory of the erythrocytes of patients with neuroacanthocytosis II
Aim of the project - Systematic analysis of the proteome (protein composition) from red cell membranes of normal and of ChAc erythrocytes (red blood cells) are carried out to identify proteins that are differently expressed. Since no data are available on systematic analysis of the red cell membrane proteome of ChAc erythrocytes, this analysis had to be carried out to evaluate whether morphological and functional differences between normal and ChAc red cells were related to quantitative and/or qualitative differences between the red cell membrane proteomes. The identified proteins are not only descriptive data, but they also indicate that the major differences between red cell membrane proteomes are related to possible post-translational modifications in the protein. In fact, the preliminary data on the phosphotyrosine proteome supported this observation.
Workplan - The analysis of the phospho-tyrosine proteome in normal and ChAC red cells as well as on MLS erythrocytes will be completed. Subsequently, the phospho-data will then be used for computational analysis of phospho-networks in order to identify novel signaling pathways to be validated in diseased red cells. This strategy will allow us to highlight new potential regulatory mechanisms to be validated as markers in disease pathophysiology. This may also be useful to clinical practice in patients’ diagnoses or follow-ups.
Associated working group: Skin fibroblasts as model cells for studying the pathophysiology of chorea-acanthocytosis
Aims of the project - This project aims to elucidate the molecular pathophysiological consequences of the chorein knock-down in skin fibroblasts from confirmed chorea-acanthocytosis (ChAc) patients. Chorein is the protein encoded by the gene mutated in chorea-acanthocytosis (VPS13A) and most likely plays a role on intracellular trafficking of proteins between the trans-Golgi network and multivesicular body/late endosome. However, the exact function of this protein and the mechanisms of its involvement in the pathogenesis of ChAc remain enigmatic.
Workplan - Since ChAc is a rare disorder, the proposed consortium provides the unique opportunity to establish fibroblast cell lines from several genetically or biochemically proven ChAc patients (control cell lines from healthy controls and patients with other neuroacanthocytosis syndromes are already established by the group of the applicant). First, the chorein knock-down will be verified on the mRNA level using quantitative RT-PCR and on the protein level using Western blotting in various cell fractions such as membranes, nucleus and cytoplasm (in cooperation with the Danek group). Then, we plan to analyze the standard cell biology properties (proliferation, cell death, apoptosis, energy demand, etc.) of ChAc fibroblasts in comparison to their wild-type counterparts using standard techniques such as BrdU staining, PI-FACS analyses, lactate production measurements etc (Hermann et al. 2004, Hermann et al. 2006). These data will provide insights into the cellular consequences of alterations of chorein expression/function on the general cell biology level.
Since there are no conclusive data on the molecular function of chorein, we plan to use both hypothesis-independent and hypothesis-driven approaches to elucidate the molecular mechanisms involved in chorein knock-down:
First, we shall perform microarray-based whole transcriptome analyses of ChAc fibroblasts compared to wild-type fibroblasts as described previously (Maisel et al. 2007) to define cellular systems altered or regulated by chorein and to generate hypothesis for both the physiological and pathophysiological functions of chorein. We will additionally use a novel strategy to determine master regulatory protein networks in MELAS syndrome by combining the classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses. This technique was recently published by the subproject leader (Mende et al. 2008).
On the other hand, the preferential dysfunction/degeneration of the striatum in ChAc patients prompts to the hypothesis that the chorein knock-down alters the cellular energy metabolism (Pulsinelli 1985). We therefore plan to analyze the cellular energy metabolism including measurements of the mitochondrial complex activities as well as the levels of intracellular adenine and pyridine nucleotides (Storch et al. 1999). All major laboratory techniques are already established in the research group of the applicant (Storch et al. 1999, Hermann et al. 2004, Hermann et al. 2006, Maisel et al. 2007). Our data will provide first insights into the function of the chorein protein as well as the pathophysiological mechanisms of ChAc particularly concerning the cellular energy metabolism.
Together, the present project aims to dissect the molecular and cellular mechanisms of neurodegeneration induced by mutations in the VPS13A gene by using skin fibroblasts as a tool. The results will provide insights into the pathogenetic processes not only of ChAc as a rare neurodegenerative disorder, but also of neurodegenerative mechanisms in general.
Associated working group: Development of new tools for the analysis of chorein and other VPS13 proteins
Aims of the project - The main objective of this project is the generation, characterisation and use of new specific antibodies against chorein and other human VPS13 proteins (Velayos-Baeza et al., 2004, Genomics 84: 536-549). Previous attempts have been made before towards this same objective, always to obtain rabbit polyclonal antibodies. Most of these attempts were done by using short peptides as an antigen, and several anti-peptides were tested in several applications. Although some of them are able to detect the protein over-expressed in culture models, they did not present a high degree of specificity, and the endogenous proteins (not over-expressed) were not detected. Only one of the available “anti-VPS13” antibodies has proven useful in some applications: the anti-chorein antibody known as “anti-chor1” was raised in rabbit after immunisation not with a peptide, but with a 300-amino acids protein fragment of chorein over-expressed and purified from bacteria (Dobson-Stone et al., 2004, Ann Neurol 56: 299-302). We decided to use small VPS13 protein fragments, over-expressed and purified from bacteria, as antigens to raise antibodies in rabbit and other species in order to develop a panel of antibodies that can be used in several applications for the detection of chorein and other VPS13 proteins.
Workplan - The phases of this project are as follows:
Design of VPS13 fragments to be over-expressed, and choice of bacterial expression vectors;
PCR-amplification of cDNA encoding these protein fragments, cloning in selected vectors, and sequence verification;
Tests of over-expression in bacteria, in several culture conditions, using small volumes;
Selection of over-expression plasmids and culture conditions for scale-up;
Purification of over-expressed protein fragments from large bacterial cultures;
Immunisation of animal(s) of choice to obtain antibodies (custom service performed by different providers);
Testing of the resulting antisera (polyclonal antibodies) or clones (monoclonal antibodies) for different applications (WB, IF, immunohistochemistry, etc).
Antonio
Velayos-Baeza, PhD (
) Anthony
P. Monaco, MD, PhD Wellcome Trust Centre for Human Genetics Oxford, United Kindgom Project Homepage: www.well.ox.ac.uk/monaco/chac.shtml
