Claus Christensen

An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons.

The Neural Cell Adhesion Molecule (NCAM) plays a crucial role in development of the central nervous system regulating cell migration, differentiation and synaptogenesis. NCAM mediates cell–cell adhesion through homophilic NCAM binding, subsequently resulting in activation of the fibroblast growth factor receptor (FGFR). NCAM‐mediated adhesion leads to activation of various intracellular signal transduction pathways, including the Ras‐mitogen activated protein kinase (MAPK) and the phosphatidylinositol‐3‐kinase (PI3K)‐Akt pathways. A synthetic peptide derived from the second fibronectin type III module of NCAM, the FGL peptide, binds to and induces phosphorylation of FGFR without prior homophilic NCAM binding. We here present evidence that this peptide is able to mimic NCAM heterophilic binding to the FGFR by inducing neuronal differentiation as reflected by neurite outgrowth through a direct interaction with FGFR in primary cultures of three different neuronal cell types all expressing FGFR subtype 1: dopaminergic, hippocampal and cerebellar granule neurons. Moreover, we show that the FGL peptide promotes neuronal survival upon induction of cell death in the same three cell types. The effects of the FGL peptide are shown to depend on activation of FGFR and the MAPK and PI3K intracellular signalling pathways, all three kinases being necessary for the effects of FGL on neurite outgrowth and neuronal survival.

Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion.

Modern microfabrication and microfluidic technologies offer new opportunities in the design and fabrication of miniaturized cell culture systems for online monitoring of living cells. We used laser micromachining and thermal bonding to fabricate an optically transparent, low-cost polymeric chip for long-term online cell culture observation under controlled conditions. The chip incorporated a microfluidic flow equalization system, assuring uniform perfusion of the cell culture media throughout the cell culture chamber. The integrated indium-tin-oxide heater and miniature temperature probe linked to an electronic feedback system created steady and spatially uniform thermal conditions with minimal interference to the optical transparency of the chip. The fluidic and thermal performance of the chip was verified by finite element modeling and by operation tests under fluctuating ambient temperature conditions. HeLa cells were cultured for up to 2 weeks within the cell culture chip and monitored using a time-lapse video recording microscopy setup. Cell attachment and spreading was observed during the first 10-20 h (lag phase). After approximately 20 h, cell growth gained exponential character with an estimated doubling time of about 32 h, which is identical to the observed doubling time of cells grown in standard cell culture flasks in a CO2 incubator.

The neural cell adhesion molecule binds to fibroblast growth factor receptor 2.

The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1–FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface plasmon resonance analysis that NCAM can bind to FGFR2 with an affinity similar to that for the NCAM–FGFR1 interaction. However, the kinetic parameters for the NCAM–FGFR2 binding are different from those of the NCAM–FGFR1 binding. Both receptors were shown to cycle relatively fast between the NCAM bound and unbound states, although FGFR2 cycling was clearly faster (13 times) than the FGFR1 cycling. Moreover, ATP was more effective in inhibiting the binding of NCAM to FGFR1 than to FGFR2, indicating that the binding sites in NCAM for the two receptors are similar, but not identical.

Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress

Both Myc and Ras oncogenes impact cellular metabolism, deregulate redox homeostasis and trigger DNA replication stress (RS) that compromises genomic integrity. However, how are such oncogene‐induced effects evoked and temporally related, to what extent are these kinetic parameters shared by Myc and Ras, and how are these cellular changes linked with oncogene‐induced cellular senescence in different cell context(s) remain poorly understood. Here, we addressed the above‐mentioned open questions by multifaceted comparative analyses of human cellular models with inducible expression of c‐Myc and H‐RasV12 (Ras), two commonly deregulated oncoproteins operating in a functionally connected signaling network. Our study of DNA replication parameters using the DNA fiber approach and time‐course assessment of perturbations in glycolytic flux, oxygen consumption and production of reactive oxygen species (ROS) revealed the following results. First, overabundance of nuclear Myc triggered RS promptly, already after one day of Myc induction, causing slow replication fork progression and fork asymmetry, even before any metabolic changes occurred. In contrast, Ras overexpression initially induced a burst of cell proliferation and increased the speed of replication fork progression. However, after several days of induction Ras caused bioenergetic metabolic changes that correlated with slower DNA replication fork progression and the ensuing cell cycle arrest, gradually leading to senescence. Second, the observed oncogene‐induced RS and metabolic alterations were cell‐type/context dependent, as shown by comparative analyses of normal human BJ fibroblasts versus U2‐OS sarcoma cells. Third, the energy metabolic reprogramming triggered by Ras was more robust compared to impact of Myc. Fourth, the detected oncogene‐induced oxidative stress was due to ROS (superoxide) of non‐mitochondrial origin and mitochondrial OXPHOS was reduced (Crabtree effect). Overall, our study provides novel insights into oncogene‐evoked metabolic reprogramming, replication and oxidative stress, with implications for mechanisms of tumorigenesis and potential targeting of oncogene addiction.

Fibroblast growth factor-derived peptides : functional agonists of the fibroblast growth factor receptor

A series of peptides, termed dekafins, were derived from the β10–β11 loop regions of fibroblast growth factors (FGFs) 1, 2, 3, 5, 6, 8, 9, 10, and 17. The dekafins share a homologous amino acid sequence similar to a sequence in the first fibronectin type III module of the neural cell adhesion molecule. All dekafins were shown by surface plasmon resonance analysis to bind fibroblast growth factor receptor (FGFR)1‐IIIc‐Ig2–3 and FGFR2‐IIIb‐Ig2–3, respectively, with Kd values of approximately 10−7 to 10−8u2003mol/L. Binding of dekafin1 to FGFR1‐IIIc‐Ig2–3 was inhibited by a heparin analog, sucrose octasulfate, indicating that heparin sulfate moiety can modulate dekafin binding to FGFRs. Treatment of transcription and mRNA export (TREX) cells permanently expressing Strep‐tag‐labeled FGFR1‐IIIc with dekafins resulted in receptor phosphorylation. FGF1‐induced FGFR1‐IIIc phosphorylation was inhibited by dekafin1 and 10 in high concentrations, indicating that dekafins are FGFR partial agonists. The dekafins induced neuronal differentiation as reflected by neurite outgrowth from cerebellar granule neurons, an effect that was abolished by SU5402, a specific inhibitor of the FGFR tyrosine kinase, and by inositolhexaphosphate, an extracellularly acting FGFR antagonist. Some, but not all, dekafins were capable of promoting survival of cerebellar granule neurons induced to undergo apoptosis. Thus, the dekafins are functional FGFR agonists with apparent therapeutic potential.

NCAM-derived peptides function as agonists for the fibroblast growth factor receptor

The neural cell adhesion molecule (NCAM) directly interacts with the fibroblast growth factor receptor (FGFR). Both fibronectin type III (FN3) modules of NCAM are involved in this interaction. One of the NCAM–FGFR contact sites has been localized recently to the upper N‐terminal part of the second NCAM FN3 module encompassing the F and G β‐strands and the interconnecting loop region. Here, we investigated whether any of the six putative strand‐loop‐strand regions in the first NCAM FN3 module are involved in FGFR interactions. Peptide sequences encompassing these regions, termed encamins, were synthesized and tested for their ability to bind and activate FGFR. Encamins localized to the N‐terminal part of the first FN3 module did not interact with FGFR, whereas encamins localized to the C‐terminal part, termed EncaminA, C and E, bound to and activated FGFR. The encamins induced FGFR‐dependent neurite outgrowth, and EncaminC and E promoted neuronal survival and enhanced pre‐synaptic function. In conclusion, the interaction between NCAM and FGFR probably involves multiple contact sites at an interface formed by the two NCAM FN3 modules and FGFR, and encamins could constitute important pharmacological tools for the study of specific functional aspects of NCAM, including neuroprotection and modulation of plasticity.

Molecular characterization of a Leishmania donovanii cDNA clone with similarity to human 20S proteasome a-type subunit

Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L. donovanii poly(A(+))mRNA indicated the isolation of a full length cDNA clone with a transcript size of 1.9 kb. The expressed recombinant LePa fusion protein induced proliferation of peripheral blood mononuclear cells in one out of seven patients who had suffered from visceral leishmaniasis. Plasma from 16 out of 25 patients with visceral leishmaniasis and four out of 18 patients with cutaneous leishmaniasis contained IgG antibodies which reacted with the purified LePa fusion protein as evaluated in an ELISA. The LePa DNA sequence was inserted into an eukaryotic expression vector and Balb/c mice were vaccinated. DNA vaccination of Balb/c mice with LePa generated an initial significant reduction in lesion size after challenge.

Polyethylenimine architecture-dependent metabolic imprints and perturbation of cellular redox homeostasis.

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.

Production of interferon-gamma and interleukin-4 by human T cells recognizing Leishmania lipophosphoglycan-associated protein

The Leishmania protein LPGAP which is co-isolated with lipophosphoglycan is a specific activator of T cells from individuals who have recovered from American leishmaniasis. We have tested the effect of LPGAP on peripheral blood mononuclear cells (PBMC) from Kenyan donors cured from L. donovani infections. LPGAP induced vigorous proliferation and production of interferon-gamma (IFN-gamma) by the cells. In addition PBMC incubated with LPGAP released interleukin-4 (IL-4) after pulsing with ionomycin and phorbol myristate acetate. Single cells were isolated from LPGAP-stimulated cell lines and expanded as T-cell clones. LPGAP-reactive T-cell clones were activated by crude preparations of both promastigotes and axenic grown amastigote-like parasites. Among 9 CD4+ T-cell clones recognizing LPGAP, cells secreting predominantly IFN-gamma as well as cells secreting predominantly IL-4 were identified. The results show that both IFN-gamma producing (Th1-like) and IL-4 producing (Th2-like) T cells recognizing LPGAP are expanded after infection with L. donovani in humans.

Agonists of fibroblast growth factor receptor induce neurite outgrowth and survival of cerebellar granule neurons.

Fibroblast growth factor receptor (FGFR) signaling is pivotal in the regulation of neurogenesis, neuronal differentiation and survival, and synaptic plasticity both during development and in adulthood. In order to develop low molecular weight agonists of FGFR, seven peptides, termed hexafins, corresponding to the β6‐β7 loop region of the FGF 1, 2, 3, 8, 9, 10, and 17, were synthesized. This region shares a homologous amino acid sequence with the FG‐loop region of the second fibronectin Type III module of the neural cell adhesion molecule (NCAM) that binds to the FGFR. Hexafins were shown by surface plasmon resonance to bind to FGFR1‐IIIc‐Ig2‐3 and FGFR2‐IIIb‐Ig2‐3. The heparin analog sucrose octasulfate inhibited hexafin binding to FGFR1‐IIIc‐Ig2‐3 indicating overlapping binding sites. Hexafin‐binding to FGFR1‐IIIc resulted in receptor phosphorylation, but inhibited FGF1‐induced FGFR1 phosphorylation, indicating that hexafins act as partial agonists. Hexafin2, 3, 8, 10, and 17 (but not 1 or 9) induced neurite outgrowth from cerebellar granule neurons (CGNs), an effect that was abolished by two inhibitors of FGFR, SU5402 and inositol hexaphosphate (IP6) and a diacylglycerol lipase inhibitor, RHC‐80267. The neuritogenic effects of selected hexafins could also be inhibited by FGF1 which by itself did not induce neurite outgrowth. Moreover, hexafin1, 3, 9, 10, and 17 (but not 2 or 8) promoted survival of CGNs induced to undergo apoptosis. Thus, selected hexafins induced neuronal differentiation and survival, making them promising pharmacological tools for the study of functional FGFR regulation in development of the nervous system.