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Klein R.,University of Cologne | Klein R.,Max Planck Institute for Neurological Research | Blaschke S.,University of Cologne | Neumaier B.,Max Planck Institute for Neurological Research | And 12 more authors.
Stem Cell Reviews and Reports | Year: 2014

The neural cell adhesion molecule (NCAM) plays a role in neurite outgrowth, synaptogenesis, and neuronal differentiation. The NCAM mimetic peptide FG Loop (FGL) promotes neuronal survival in vitro and enhances spatial learning and memory in rats. We here investigated the effects of FGL on neural stem cells (NSC) in vitro and in vivo. In vitro, cell proliferation of primary NSC was assessed after exposure to various concentrations of NCAM or FGL. The differentiation potential of NCAM- or FGL-treated cells was assessed immunocytochemically. To investigate its influence on endogenous NSC in vivo, FGL was injected subcutaneously into adult rats. The effects on NSC mobilization were studied both via non-invasive positron emission tomography (PET) imaging using the tracer [18F]-fluoro-l-thymidine ([18F]FLT), as well as with immunohistochemistry. Only FGL significantly enhanced NSC proliferation in vitro, with a maximal effect at 10 μg/ml. During differentiation, NCAM promoted neurogenesis, while FGL induced an oligodendroglial phenotype; astrocytic differentiation was neither affected by NCAM or FGL. Those differential effects of NCAM and FGL on differentiation were mediated through different receptors. After FGL-injection in vivo, proliferative activity of NSC in the subventricular zone (SVZ) was increased (compared to placebo-treated animals). Moreover, non-invasive imaging of cell proliferation using [18F]FLT-PET supported an FGL-induced mobilization of NSC from both the SVZ and the hippocampus. We conclude that FGL robustly induces NSC mobilization in vitro and in vivo, and supports oligodendroglial differentiation. This capacity renders FGL a promising agent to facilitate remyelinization, which may eventually make FGL a drug candidate for demyelinating neurological disorders. © Springer Science+Business Media New York 2014.


Xu R.,ENKAM Pharmaceuticals A S | Xu R.,University of Aarhus | Srinivasan S.P.,University of Cologne | Sureshkumar P.,University of Cologne | And 7 more authors.
Cellular Physiology and Biochemistry | Year: 2015

Background/Aims: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. Methods: In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGLL, hNgf-C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. Results: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. Conclusion: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired. © 2015 S. Karger AG, Basel.


Xu R.,ENKAM Pharmaceuticals A S | Xu R.,University of Geneva | Feyeux M.,University of Geneva | Julien S.,University of Geneva | And 6 more authors.
AAPS Journal | Year: 2014

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons. © 2014 American Association of Pharmaceutical Scientists.


Kern I.,University of Geneva | Xu R.,ENKAM Pharmaceuticals A S | Xu R.,University of Geneva | Julien S.,University of Geneva | And 8 more authors.
Current Medicinal Chemistry | Year: 2013

Neural differentiation of embryonic stem cells (ESC) is considered a promising model to perform in vitro testing for neuroactive and neurotoxic compounds. We studied the potential of a dual reporter murine ESC line to identify bioactive and/or toxic compounds. This line expressed firefly luciferase under the control of the neural cell-specific tubulin alpha promoter (TUBA1A), and renilla luciferase under the control of the ubiquitous translation elongation factor 1-Alpha-1 (EEF1A1) promoter. During neural differentiation, TUBA1A activity increased, while EEF1A1 activity decreased. We first validated our test system using the known neurotoxin methyl mercury. This compound altered expression of both reporter genes, with ESC-derived neural precursors being affected at markedly lower concentrations than undifferentiated ESCs. Analysis of a library of 1040 bioactive compounds picked up 127 compounds with altered EEF1A1 and/or TUBA1A promoter activity, which were classified in 4 clusters. Cluster 1 (low EEF1A1 and TUBA1A) was the largest cluster, containing many cytostatic drugs, as well as known neurodevelopmental toxicants, psychotropic drugs and endocrine disruptors. Cluster 2 (high EEF1A1, stable TUBA1A) was limited to three sulfonamides. Cluster 3 (high EEF1A1 and TUBA1A) was small, but markedly enriched in neuroactive and neurotoxic compounds. Cluster 4 (stable EEF1A1, high TUBA1A) was heterogeneous, containing endocrine disruptors, neurotoxic and cytostatic drugs. The dual reporter gene assay described here might be a useful addition to in vitro drug testing panels. Our two-dimensional testing strategy provides information on complex response patterns, which could not be achieved by a single marker approach. © 2013 Bentham Science Publishers.


Grant
Agency: Cordis | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2010-ITN | Award Amount: 3.76M | Year: 2011

Protein aggregation is a hallmark of many late onset neurodegenerative disorders including Parkinsons Disease (PD), Alzheimers Disease (AD), amyotrophic lateral sclerosis (ALS), prion diseases as well as the group of polyglutamine diseases (polyQ). The aim of this proposal is to create a network of European partners bridging important basic mechanisms involved in proteinopathies, research of model diseases and treatment approaches. The TreatPolyQ network will focus on two main representatives of the polyQ diseases: Huntingtons disease as the most common polyQ disease as well as spinocerebellar ataxia type 3 (SCA3) as the most frequent autosomal-dominantly inherited ataxia. Patients suffer from a multitude of neurological symptoms including movement abnormalities with late onset and in a progressive manner. Up to now, no treatment or cure is available. The network will be consisting of a rare combination of experts from basic and translational research, including a Nobel prize laureate, four industrial partners (two medium, and two small companies, all incorporated as full participants) and academic leaders of the field. The network not only focuses on one special aspect of a disease but spans several important disease-associated mechanism as well as promising treatment strategies for HD and SCA3 (protein transport, protein folding, protein degradation via both the ubiquitin-proteasome system and autophagy), likely to be important across a range of neurodegenerative diseases. In order to implement these research projects, extensive collaborations and temporarily personnel secondments of the involved researchers will take place, enhancing interdisciplinary transfer of knowledge. Beyond the personalized local training plan for each employed researcher within the Network, there will be 4 structured courses covering aspects ranging from structural biology to protein degradation to model organisms and drug development, including soft skill training.


Grant
Agency: Cordis | Branch: FP7 | Program: MC-IAPP | Phase: FP7-PEOPLE-2009-IAPP | Award Amount: 1.49M | Year: 2010

The STEMCAM project is a 4-years training and transfer of knowledge program between two distinguished academic groups in stem cell research, two highly innovative SMEs and a company leader in development of media for stem cell research, to foster long-term industry-academy collaboration and partnership in the field of stem cells research and applications. The scientific and industrial aim of STEMCAM is to study the role of the Neuronal Cell Adhesion Molecule NCAM and related growth factors in the maintenance, survival and differentiation of induced pluripotent stem (iPS) cells towards the neural and myocardial lineage, in comparison with embryonic stem cells. The project will take advantage of very unique and innovative pharmacological tools, the NCAM and growth factor mimetic peptides discovered by ENKAM. To achieve its aim STEMCAM will apply an interdisciplinary approach from cell biology (including innovative in vitro culture using bi- and three dimensional systems), immunocytochemistry, imaging, molecular biology, electrophysiology, to peptide chemistry and chemioinformatics. The project will run via a training and transfer of knowledge program structured to efficiently exploit the expertise and complementarities between the industrial and academic partners to reach the scientific goals of the project and provide high quality intersectorial training for the participating researchers. This is expected to be of high benefit for their individual career development. The STEMCAM project links intersectorial research activities in two very relevant areas of stem cell research, neurogenesis and cardiomyogenesis and will strictly collaborate with the IAPPs INDUSTEM and PARTNERS and the large FP7 IP ESNATS, complementing and expanding with its unique approach their research scope and transfer of knowledge. Thus STEMCAM will significantly contribute to progress of stem cell research in Europe with high potential impact on European competitiveness and regenerative medicine


Grant
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2011.1.4-1 | Award Amount: 7.81M | Year: 2012

Neurodegenerative disorders such as, Alzheimers disease (AD), Mild Cognitive Impairment (MCI), stroke, Traumatic Brain Injury (TBI) and chronic stress create a major economic burden to society and a substantial reduction in quality of life for patients and families. The development of neuroregenerative therapies is notoriously difficult and requires significant investment. NeuroFGL will contribute to decrease these barriers through: (1) the clinical advancement of a promising novel regenerative therapy (FGLs) for neurological disorders, (2) hedging the clinical development by developing tests that enable early clinical assessments to be made, thereby maximising the chance that FGL and other neurogenerative therapies actually become developed to the benefit of patients and society; and (3) Selecting a target patient population with less variability and thereby easier to study reducing and time resources needed, and increase predictability . FGLs is a promising and novel regenerative therapy being the clinical lead development candidate selected from a group of allosteric FGF-receptor modulators (referred to as FGL) mimicking NCAM. FGL has demonstrated positive effects in a number of in vivo models of neurodegeneration, e.g. beta-amyloid induced toxicity, global ischemia and chronic stress. The in vivo effects of FGL suggest a disease-modifying activity in several neurodegenerative disorders, such as neurogenesis. A phase I clinical study has demonstrated a FGL peptide to be well tolerated and safe. NeuroFGL will refine existing and develop new tests and techniques, that will at an early stage of the clinical development: (1) provide better information on the mechanisms of action (NCAM mimicking allosteric FGF recoter modulation) in man, (2) deliver translational effects seen between animal and man, (3) provide results earlier and cheaper, increasing the iteratiation and (4) select patients with conditions associated with less variability, e.g. patients with AD with a specific EEG or patients progressing to AD identified in patients with MCI. These developments will together provide a more robust basis for the development of FGLs, other drugs with a similar mechanism of action and other therapies for neurodegenerative disorders.


Kohler L.B.,ENKAM Pharmaceuticals A S | Kohler L.B.,Copenhagen University | Soroka V.,Copenhagen University | Korshunova I.,ENKAM Pharmaceuticals A S | And 3 more authors.
Journal of Neuroscience Research | Year: 2010

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N-terminal immunoglobulin (Ig)-like modules indicates that the first and second Ig modules bind to each other, thereby presumably mediating dimerization of NCAM molecules expressed on the same cell surface (cis-interactions), whereas the third Ig module, through interactions with the first or second Ig module, mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans-interactions). We have designed a new potent peptide ligand of NCAM, termed plannexin, based on a discontinuous sequence in the second NCAM Ig module that represents a homophilic binding site for an opposing third Ig module. The peptide was found by surface plasmon resonance analysis to bind the third NCAM Ig module. It promoted survival of cultured cerebellar granule neurons (CGNs) and also induced neurite extension in cultures of dopaminergic neurons and CGNs; the latter effect was shown to be dependent on NCAM expression, indicating that plannexin mimics the neuritogenic effect of homophilic NCAM binding. © 2010 Wiley-Liss, Inc.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH-2007-2.2.1-4 | Award Amount: 4.14M | Year: 2008

Memory loss is a central symptom in different diseases, and represents a significant social and economic burden for a large percentage of European citizens. The molecular and neurobiological bases of memory deficits are largely unknown and there are currently no drugs available that can markedly decelerate or prevent memory decline. To address this major problem, this project will investigate the role of novel synaptic cell adhesion molecules (CAMs) in memory loss, and the therapeutic value of targeting these CAMs to restore memory function and associated neurobiological mechanisms at the synaptic level. The selection of novel synaptic CAMs is based on (1) recent evidence showing that they can recruit the synaptic machinery and form synapses; (2) the implication of CAMs, in general, in synaptic plasticity and memory, and (3) a direct link established between synaptic remodelling and memory formation. We have assembled a multidisciplinary consortium including excellent European researchers in this field, covering a wide range of structural, biochemical, electrophysiological and behavioural expertise. To understand memory loss, three domains have been selected as among the most prominent and widespread disease domains affecting memory and quality of life in our society: psychiatric disorders, the neurodegenerative disorder of Alzheimer's disease, stress and aging. For each disease domain, specific and validated animal models will be used to investigate memory loss on molecular, subcellular, cellular and functional levels. Our approach will result in the preclinical development and validation of mimetic peptides for novel synaptic CAMs as potential drug candidates to treat memory deficits or prevent memory decline. The current proposal offers the groundbreaking possibility to raise the bar in memory research by targeting novel molecules and developing effective drugs to treat memory disturbances based on their biological mechanism.


PubMed | ENKAM Pharmaceuticals A S
Type: Journal Article | Journal: The AAPS journal | Year: 2014

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons.

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