Assessing Human Embryonic Stem Cell-Derived Dopaminergic Neuron Progenitor Transplants Using Non-invasive Imaging Techniques.

PURPOSE: Human pluripotent stem cell (hPSC)-derived dopaminergic neuron progenitor cells (DAPCs) are a potential therapy for Parkinson's disease (PD). However, their intracranial administration raises safety concerns including uncontrolled proliferation, migration and inflammation. Here, we apply a bimodal imaging approach to investigate the fate of DAPC transplants in the rat striatum. PROCEDURES: DAPCs co-expressing luciferase and ZsGreen or labelled with micron-sized particles of iron oxide (MPIOs) were transplanted in the striatum of RNU rats (n = 6 per group). DAPCs were tracked in vivo using bioluminescence and magnetic resonance (MR) imaging modalities. RESULTS: Transgene silencing in differentiating DAPCs accompanied with signal attenuation due to animal growth rendered the bioluminescence undetectable by week 2 post intrastriatal transplantation. However, MR imaging of MPIO-labelled DAPCs showed that transplanted cells remained at the site of injection for over 120 days. Post-mortem histological analysis of DAPC transplants demonstrated that labelling with either luciferase/ZsGreen or MPIOs did not affect the ability of cells to differentiate into mature dopaminergic neurons. Importantly, labelled cells did not elicit increased glial reactivity compared to non-labelled cells. CONCLUSIONS: In summary, our findings support the transplantation of hPSC-derived DAPCs as a safe treatment for PD.

Impulsive choices in mice lacking imprinted Nesp55.

Genomic imprinting is the process whereby germline epigenetic events lead to parent-of-origin specific monallelic expression of a number of key mammalian genes. The imprinted gene Nesp is expressed from the maternal allele only and encodes for Nesp55 protein. In the brain, Nesp55 is found predominately in discrete areas of the hypothalamus and midbrain. Previously, we have shown that loss of Nesp55 gives rise to alterations in novelty-related behaviour. Here, we extend these findings and demonstrate, using the Nespm/+ mouse model, that loss of Nesp55 leads to impulsive choices as measured by a delayed-reinforcement task, whereby Nespm/+ mice were less willing to wait for a delayed, larger reward, preferring instead to choose an immediate, smaller reward. These effects were highly specific as performance in another component of impulsive behaviour, the ability to stop a response once started as assayed in the stop-signal reaction time task, was equivalent to controls. We also showed changes in the serotonin system, a key neurotransmitter pathway mediating impulsive behaviour. First, we demonstrated that Nesp55 is co-localized with serotonin and then went on to show that in midbrain regions there were reductions in mRNA expression of the serotonin-specific genes Tph2 and Slc6a4, but not the dopamine-specific gene Th in Nespm/+ mice; suggesting an altered serotonergic system could contribute, in part, to the changes in impulsive behaviour. These data provide a novel mode of action for genomic imprinting in the brain and may have implications for pathological conditions characterized by maladaptive response control.

Imprinting the Gnas locus.

Gnas is an enigmatic and rather complex imprinted gene locus. A single transcription unit encodes three, and possibly more, distinct proteins. These are determined by overlapping transcripts from alternative promoters with different patterns of imprinting. The canonical Gnas transcript codes for Gsalpha, a highly conserved signalling protein and an essential intermediate in growth, differentiation and homeostatic pathways. Monoallelic expression of Gnas is highly tissue-restricted. The alternative transcripts encode XLalphas, an unusual variant of Gsalpha, and the chromogranin-like protein Nesp55. These transcripts are expressed specifically from the paternal and maternal chromosomes, respectively. Their existence in the Gnas locus might imply functional connections amongst them or with Gsalpha. In this review, we consider how imprinting of Gnas was discovered, the phenotypic consequences of mutations in each of the gene products, both in the mouse and human, and provide some conjectures to explain why this elaborate imprinted locus has evolved in this manner in mammals.

The contactin-related protein FAR-2 defines purkinje cell clusters and labels subpopulations of climbing fibers in the developing cerebellum.

FAR-2 is a novel neural member of the Ig superfamily, which is related to F11/F3/contactin and axonin-1/TAG-1. This protein is expressed by subpopulations of Purkinje cells in the chicken cerebellum and FAR-2-positive clusters of these neurons alternate with FAR-2-negative clusters in both tangential dimensions of the cerebellar cortex. Furthermore, FAR-2 is also expressed by one type of Purkinje cell afferents, namely, the climbing fibers, and different subpopulations of these axons show distinct levels of FAR-2 expression. Homology modeling using axonin-1 as a template reveals that the four aminoterminal Ig domains of FAR-2 form a compact U-shaped structure, which is likely to contain functionally important ligand-binding sites. FAR-2 is binding to the Ig superfamily protein NgCAM/L1, but not to the related receptor NrCAM, and it is also interacting with the modular ECM protein tenascin-R. These results suggest that FAR-2 may contribute to the formation of somatotopic maps of cerebellar afferents during the development of the nervous system.

A new module arrangement for plasmapheresis.

On-line plasmapheresis using microporous membranes for filtration normally requires two external circulations (double treatment) before the detoxified plasma can be returned to the patient. The duomodule, a new filter arrangement developed by our group, integrates both steps in one equipment module using only one external circuit. Separations of aqueous polyethylene glycol (PEG) solutions as well as human plasma were carried out using the duomodule arrangement. The results revealed a considerable decrease of higher molecular substances in the feed solutions and a significant increase of these components in the permeate solution accumulated in the external compartment of the module. In conclusion, the duomodule arrangement seems to be an useful tool for the therapeutic apheresis.

Neurotractin, a novel neurite outgrowth-promoting Ig-like protein that interacts with CEPU-1 and LAMP.

The formation of axon tracts in nervous system histogenesis is the result of selective axon fasciculation and specific growth cone guidance in embryonic development. One group of proteins implicated in neurite outgrowth, fasciculation, and guidance is the neural members of the Ig superfamily (IgSF). In an attempt to identify and characterize new proteins of this superfamily in the developing nervous system, we used a PCR-based strategy with degenerated primers that represent conserved sequences around the characteristic cysteine residues of Ig-like domains. Using this approach, we identified a novel neural IgSF member, termed neurotractin. This GPI-linked cell surface glycoprotein is composed of three Ig-like domains and belongs to the IgLON subgroup of neural IgSF members. It is expressed in two isoforms with apparent molecular masses of 50 and 37 kD, termed L-form and S-form, respectively. Monoclonal antibodies were used to analyze its biochemical features and histological distribution. Neurotractin is restricted to subsets of developing commissural and longitudinal axon tracts in the chick central nervous system. Recombinant neurotractin promotes neurite outgrowth of telencephalic neurons and interacts with the IgSF members CEPU-1 (KD = 3 x 10(-8) M) and LAMP. Our data suggest that neurotractin participates in the regulation of neurite outgrowth in the developing brain.

The gene of the neural cell recognition molecule F11: conserved exon-intron arrangement in genes of neural members of the immunoglobulin superfamily.

The chicken neural glycoprotein F11 is a cell recognition molecule implicated in neurohistogenesis, in particular in the context of neurite outgrowth and fasciculation. F11 is a glycosyl-phosphatidylinositol-linked member of the immunoglobulin superfamily that is also termed contactin or F3 in humans and rodents, respectively. In this study, we report the complete structure of the F11 gene. It is composed of 23 exons distributed over more than 100 kb of genomic DNA and each of the ten domains of the F11 protein is encoded by two exons. The sizes of the introns vary by two orders of magnitude ranging from 150 bp to more than 15 kb. All interdomain introns are in phase one, i.e. are inserted after the first nucleotide of a codon, being consistent with assembly of a F11 progenitor gene via exon shuffling. The intradomain introns are localized at variable sites within the domains and have different intron phases. This study reveals a remarkable similarity of the F11 gene with the gene of axonin-1, a related neural immunoglobulin superfamily member which is also implicated in neurite outgrowth and fasciculation. The intron positions with respect to the protein domain organization are found to be identical, strongly suggesting that both genes are derived from a common ancestor that already had this exon-intron structure.