Vascular endothelial growth factor C acts as a neurotrophic factor for dopamine neurons in vitro and in vivo.

Neurotrophic factors regulate the development and maintenance of the nervous system and protect and repair dopaminergic neurons in animal models of Parkinson's disease (PD). Vascular endothelial growth factors A (VEGF-A) and B have also neurotrophic effects on various types of neurons, including dopaminergic neurons. We examined the ability of the key lymphangiogenic factor VEGF-C to protect dopaminergic cells in vitro and in vivo. The study was initiated by a finding from microarray profiling of Neuro2A-20 cells which revealed up-regulation of VEGF-C by glial cell-line-derived neurotrophic factor (GDNF). Next, we observed that VEGF-C can rescue embryonic dopaminergic neurons and activate the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway in vivo. VEGF receptors 1-2 and co-receptors, neuropilins 1-2, were expressed both in mouse embryonic cultures and adult midbrains. In vivo, VEGF-C had a robust functional effect in the rat unilateral 6-hydroxydopamine (6-OHDA) model of PD and there was a small additive effect on the survival of tyrosine hydroxylase (TH)-positive cells with GDNF. The neuroprotective effect of VEGF-C is most likely due to a combination of direct and indirect neurotrophic effects because, VEGF-C, unlike GDNF, induced also angiogenesis in the striatum following 6-OHDA insult as it did in human umbilical vein endothelial cells (HUVEC). However, we detected activation of astroglia and microglia as well as blood-brain barrier disruption after intracerebral delivery of VEGF-C, raising a concern of its safe usage as a therapeutic molecule. Our results provide evidence of VEGF-C as a neurotrophic factor that influences the dopaminergic system through multiple mechanisms.

Medium-throughput computer aided micro-island method to assay embryonic dopaminergic neuron cultures in vitro.

In Parkinson's disease (PD) midbrain dopaminergic (DA) neurons degenerate and die, causing loss of motor function. Currently no therapies exist to ameliorate neurodegeneration or to restore DA neurons, although neurotrophic factors (NTFs) are promising leads. Prior in vivo studies the NTFs are routinely assessed in vitro by quantifying the survival of DA neurons from embryonic rodent midbrain cultures. Current in vitro methods are limited in terms of assay reliability, arduous workflow, low throughput, low statistical power and may obscure detection of molecules with minor yet critically important therapeutic effects. We have developed a medium-throughput, micro-island culture method. It permits analysis of 10-12 data points from a single embryo - several fold more than any previously published method - and enables comparisons of DA neurons from a single gene knockout (KO) embryo. It is computer-aided, improves statistical power and decreases the number of animals and workload per experiment. This method enhances testing capabilities of NTFs and other factors, and enables small scale screening of chemical drug libraries. We have validated the method by confirming the known effects of glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and demonstrated additive effects via simultaneous addition of GDNF and heparin binding growth associated molecule (HB-GAM). We also show for the first time that DA neurons isolated from GDNF receptor RET-deficient mice are still GDNF responsive, suggesting the presence of an alternative non-RET receptor for GDNF in the DA system. Finally, the method can be adapted for analyses of other low abundance neuronal systems.

Purification and characterization of the assembly factor P17 of the lipid-containing bacteriophage PRD1.

Assembly factors, proteins assisting the formation of viral structures, have been found in many viral systems. The gene encoding the assembly factor P17 of bacteriophage PRD1 has been cloned and expressed in Escherichia coli. P17 acts late in phage assembly, after capsid protein folding and multimerization, and sorting of membrane proteins has occurred. P17 has been purified to near homogeneity. It is a tetrameric protein displaying a rather high heat stability. The protein is largely in an alpha-helical conformation and possesses a putative leucine zipper which is not essential for protein function, as judged by in vitro mutagenesis and complementation analysis. Although heating does not cause structural changes in the conformation of the protein, the dissociation of the tetramer into smaller units is evident as diminished self-quenching of the fluorescently labeled P17. Similarly, dissociation of the tetramer is also obtained by dialysis of the protein against 6-M guanidine hydrochloride (GdnHCl) or 1% SDS. The reassembly of these smaller units upon cooling is evident from resonance energy transfer.

Expression in Escherichia coli of rat neurotensin receptor fused to membrane proteins from the membrane-containing bacteriophage PRD1.

Bacteriophage PRD1 is a membrane-containing phage which could be used for expression of foreign membrane proteins such as neurotensin receptor (NTR), a seven-helix G-protein coupled receptor. To ensure recognition of NTR by the phage system six different fusion genes were constructed, each encoding a different phage integral membrane protein fused to the N-terminus of NTR, and expression of the fusion proteins in Escherichia coli was analysed. Here we report the identification of two fusion constructs that retained the function of NTR in E. coli. This provides the basis to develop the phage system as a heterologous expression system for seven-helix receptors.

Genome organization of membrane-containing bacteriophage PRD1.

We have determined the nucleotide sequence of the late region (11 kbp) of the lipid-containing bacteriophage PRD1. Gene localization was carried out by complementing nonsense phage mutants with genomic clones containing specific reading frames. The localization was confirmed by sequencing the N-termini of isolated gene products as well as sequencing the N-termini of tryptic fragments of the phage membrane-associated proteins. This, with the previously obtained sequence of the early regions, allowed us to organize most of the phage genes in the phage genome.