Carsten W. Lederer

Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal disorder caused by the progressive degeneration of motoneurons in brain and spinal cord. Despite identification of disease-linked mutations, the diversity of processes involved and the ambiguity of their relative importance in ALS pathogenesis still represent a major impediment to disease models as a basis for effective therapies. Moreover, the human motor cortex, although critical to ALS pathology and physiologically altered in most forms of the disease, has not been screened systematically for therapeutic targets.ResultsBy whole-genome expression profiling and stringent significance tests we identify genes and gene groups de-regulated in the motor cortex of patients with sporadic ALS, and interpret the role of individual candidate genes in a framework of differentially expressed pathways. Our findings emphasize the importance of defense responses and cytoskeletal, mitochondrial and proteasomal dysfunction, reflect reduced neuronal maintenance and vesicle trafficking, and implicate impaired ion homeostasis and glycolysis in ALS pathogenesis. Additionally, we compared our dataset with publicly available data for the SALS spinal cord, and show a high correlation of changes linked to the diseased state in the SALS motor cortex. In an analogous comparison with data for the Alzheimers disease hippocampus we demonstrate a low correlation of global changes and a moderate correlation for changes specifically linked to the SALS diseased state.ConclusionGene and sample numbers investigated allow pathway- and gene-based analyses by established error-correction methods, drawing a molecular portrait of the ALS motor cortex that faithfully represents many known disease features and uncovers several novel aspects of ALS pathology. Contrary to expectations for a tissue under oxidative stress, nuclear-encoded mitochondrial genes are uniformly down-regulated. Moreover, the down-regulation of mitochondrial and glycolytic genes implies a combined reduction of mitochondrial and cytoplasmic energy supply, with a possible role in the death of ALS motoneurons. Identifying candidate genes exclusively expressed in non-neuronal cells, we also highlight the importance of these cells in disease development in the motor cortex. Notably, some pathways and candidate genes identified by this study are direct or indirect targets of medication already applied to unrelated illnesses and point the way towards the rapid development of effective symptomatic ALS therapies.

Bone marrow as an alternative site for islet transplantation

The liver is the current site for pancreatic islet transplantation, but has many drawbacks due to immunologic and nonimmunologic factors. We asked whether pancreatic islets could be engrafted in the bone marrow (BM), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. Syngeneic islets engrafted efficiently in the BM of C57BL/6 mice rendered diabetic by streptozocin treatment. For more than 1 year after transplantation, these animals showed parameters of glucose metabolism that were similar to those of nondiabetic mice. Islets in BM had a higher probability to reach euglycemia than islets in liver (2.4-fold increase, P = .02), showed a compact morphology with a conserved ratio between alpha and beta cells, and affected bone structure only very marginally. Islets in BM did not compromise hematopoietic activity, even when it was strongly induced in response to a BM aplasia-inducing infection with lymphocytic choriomeningitis virus. In conclusion, BM is an attractive and safe alternative site for pancreatic islet transplantation. The results of our study open a research line with potentially significant clinical impact, not only for the treatment of diabetes, but also for other diseases amenable to treatment with cellular transplantation.

IthaGenes: An Interactive Database for Haemoglobin Variations and Epidemiology

Inherited haemoglobinopathies are the most common monogenic diseases, with millions of carriers and patients worldwide. At present, we know several hundred disease-causing mutations on the globin gene clusters, in addition to numerous clinically important trans-acting disease modifiers encoded elsewhere and a multitude of polymorphisms with relevance for advanced diagnostic approaches. Moreover, new disease-linked variations are discovered every year that are not included in traditional and often functionally limited locus-specific databases. This paper presents IthaGenes, a new interactive database of haemoglobin variations, which stores information about genes and variations affecting haemoglobin disorders. In addition, IthaGenes organises phenotype, relevant publications and external links, while embedding the NCBI Sequence Viewer for graphical representation of each variation. Finally, IthaGenes is integrated with the companion tool IthaMaps for the display of corresponding epidemiological data on distribution maps. IthaGenes is incorporated in the ITHANET community portal and is free and publicly available at http://www.ithanet.eu/db/ithagenes.

Differential expression of molecular motors in the motor cortex of sporadic ALS

The molecular mechanisms underlying the selective neurodegeneration of motor neurons in amyotrophic lateral sclerosis (ALS) are inadequately understood. Recent breakthroughs have implicated impaired axonal transport, mediated by molecular motors, as a key element for disease onset and progression. The current work identifies the expression of 15 kinesin-like motors in healthy human motor cortex, including three novel isoforms. Our comprehensive quantitative mRNA analysis in control and sporadic ALS (SALS) motor cortex specimens detects SALS-specific down-regulation of KIF1Bbeta and novel KIF3Abeta, two isoforms we show to be enriched in the brain, and also of SOD1, a key enzyme linked to familial ALS. This is accompanied by a marked reduction of KIF3Abeta protein levels. In the motor cortex KIF3Abeta localizes in cholinergic neurons, including upper motor neurons. No mutations causing splicing defects or altering protein-coding sequences were identified in the genes of the three proteins. The present study implicates two motor proteins as possible candidates in SALS pathology.

Motor protein KIFC5A interacts with Nubp1 and Nubp2, and is implicated in the regulation of centrosome duplication.

Inhibition of motor protein activity has been linked with defects in the formation of poles in the spindle of dividing cells. However, the molecular mechanisms underlying the functional relationship between motor activity and centrosome dynamics have remained uncharacterised. Here, we characterise KIFC5A, a mouse kinesin-like protein that is highly expressed in dividing cells and tissues, and is subject to developmental and cell-type-specific regulation. KIFC5A is a minus-end-directed, microtubule-dependent motor that produces velocities of up to 1.26 μm minute-1 in gliding assays and possesses microtubule bundling activity. It is nuclear in interphase, localises to the centre of the two microtubule asters at the beginning of mitosis, and to spindle microtubules in later mitotic phases. Overexpression of KIFC5A in mouse cells causes the formation of aberrant, non-separated microtubule asters and mitotic arrest in a prometaphase-like state. KIFC5A knockdown partly rescues the phenotype caused by inhibition of plus-end-directed motor Eg5 by monastrol on the mitotic spindle, indicating that it is involved in the balance of forces determining bipolar spindle assembly and integrity. Silencing of KIFC5A also results in centrosome amplification detectable throughout the cell cycle. Supernumerary centrosomes arise primarily as a result of reduplication and partly as a result of cytokinesis defects. They contain duplicated centrioles and have the ability to organise microtubule asters, resulting in the formation of multipolar spindles. We show that KIFC5A interacts with nucleotide-binding proteins 1 and 2 (Nubp1 and Nubp2), which have extensive sequence similarity to prokaryotic division-site-determining protein MinD. Nubp1 and Nubp2 also interact with each other. Knockdown of Nubp1 or double knockdown of Nubp1 and Nubp2 (Nubp1&Nubp2) both phenocopy the KIFC5A silencing effect. These results implicate KIFC5A and the Nubp proteins in a common regulatory pathway involved in the control of centrosome duplication in mammalian cells.

Recent trends in the gene therapy of β-thalassemia.

The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases.

Plerixafor+G-CSF–mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy

Globin gene therapy requires abundant numbers of highly engraftable, autologous hematopoietic stem cells expressing curative levels of β-globin on differentiation. In this study, CD34+ cells from 31 thalassemic patients mobilized with hydroxyurea+granulocyte colony-stimulating factor (G-CSF), G-CSF, Plerixafor, or Plerixafor+G-CSF were transduced with the TNS9.3.55 β-globin lentivector and compared for transducibility and globin expression in vitro, as well as engraftment potential in a xenogeneic model after partial myeloablation. Transduction efficiency and vector copy number (VCN) averaged 48.4 ± 2.8% and 1.91 ± 0.04, respectively, whereas expression approximated the one-copy normal β-globin output. Plerixafor+G-CSF cells produced the highest β-globin expression/VCN. Long-term multilineage engraftment and persistent VCN and vector expression was encountered in all xenografted groups, with Plerixafor+G-CSF-mobilized cells achieving superior short-term engraftment rates, with similar numbers of CD34+ cells transplanted. Overall, Plerixafor+G-CSF not only allows high CD34+ cell yields but also provides increased β-globin expression/VCN and enhanced early human chimerism under nonmyeloablative conditions, thus representing an optimal graft for thalassemia gene therapy.

hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: Structural and functional studies

Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. Despite its intriguing structure, unique properties and cellular localization, the enzymatic mechanism and biological function of hCINAP have remained poorly characterized. Here, we offer the first high‐resolution structure of hCINAP in complex with the substrate ADP (and dADP), the structure of hCINAP with a sulfate ion bound at the AMP binding site, and the structure of the ternary complex hCINAP‐Mg2+ADP‐Pi. Induced fit docking calculations are used to predict the structure of the hCINAP‐Mg2+ATP‐AMP ternary complex. Structural analysis suggested a functional role for His79 in the Walker B motif. Kinetic analysis of mutant hCINAP‐H79G indicates that His79 affects both AK and ATPase catalytic efficiency and induces homodimer formation. Finally, we show that in vivo expression of hCINAP‐H79G in human cells is toxic and drastically deregulates the number and appearance of CBs in the cell nucleus. Our findings suggest that hCINAP may not simply regulate nucleotide homeostasis, but may have broader functionality, including control of CB assembly and disassembly in the nucleus of human cells. Proteins 2012;.

An Electronic Infrastructure for Research and Treatment of the Thalassemias and Other Hemoglobinopathies: The Euro-Mediterranean Ithanet Project

Hemoglobin (Hb) disorders are common, potentially lethal monogenic diseases, posing a global health challenge. With worldwide migration and intermixing of carriers, demanding flexible health planning and patient care, hemoglobinopathies may serve as a paradigm for the use of electronic infrastructure tools in the collection of data, the dissemination of knowledge, the harmonization of treatment, and the coordination of research and preventive programs. ITHANET, a network covering thalassemias and other hemoglobinopathies, comprises 26 organizations from 16 countries, including non-European countries of origin for these diseases (Egypt, Israel, Lebanon, Tunisia and Turkey). Using electronic infrastructure tools, ITHANET aims to strengthen cross-border communication and data transfer, cooperative research and treatment of thalassemia, and to improve support and information of those affected by hemoglobinopathies. Moreover, the consortium has established the ITHANET Portal, a novel web-based instrument for the dissemination of information on hemoglobinopathies to researchers, clinicians and patients. The ITHANET Portal is a growing public resource, providing forums for discussion and research coordination, and giving access to courses and databases organized by ITHANET partners. Already a popular repository for diagnostic protocols and news related to hemoglobinopathies, the ITHANET Portal also provides a searchable, extendable database of thalassemia mutations and associated background information. The experience of ITHANET is exemplary for a consortium bringing together disparate organizations from heterogeneous partner countries to face a common health challenge. The ITHANET Portal as a web-based tool born out of this experience amends some of the problems encountered and facilitates education and international exchange of data and expertise for hemoglobinopathies.

A Minimal Set of SNPs for the Noninvasive Prenatal Diagnosis of β-Thalassaemia

β‐thalassaemia is one of the commonest autosomal recessive single‐gene disorders worldwide. Prenatal tests use invasive methods, posing a risk for the pregnancy itself. Development of a noninvasive prenatal diagnostic method is, therefore, of paramount importance. The aim of the present study is to identify high‐heterozygote informative single‐nucleotide polymorphisms (SNPs), suitable for the development of noninvasive prenatal diagnosis (NIPD) of β‐thalassaemia. SNP genotyping analysis was performed on 75 random samples from the Cypriot population for 140 SNPs across the β‐globin cluster. Shortlisted, highly heterozygous SNPs were then examined in 101 carrier families for their applicability in the noninvasive detection of paternally inherited alleles. Forty‐nine SNPs displayed more than 6% heterozygosity and were selected for NIPD analysis, revealing 72.28% of the carrier families eligible for qualitative SNP‐based NIPD, and 92% for quantitative detection. Moreover, inference of haplotypes showed predominant haplotypes and many subhaplotypes with sufficient prevalence for diagnostic exploitation. SNP‐based analyses are sensitive and specific for the detection of the paternally inherited allele in maternal plasma. This study provides proof of concept for this approach, highlighting its superiority to NIPD based on single markers and thus providing a blueprint for the general development of noninvasive prenatal diagnostic assays for β‐thalassaemia.