Disruption of myelin leads to ectopic expression of K(V)1.1 channels with abnormal conductivity of optic nerve axons in a cuprizone-induced model of demyelination.

The molecular determinants of abnormal propagation of action potentials along axons and ectopic conductance in demyelinating diseases of the central nervous system, like multiple sclerosis (MS), are poorly defined. Widespread interruption of myelin occurs in several mouse models of demyelination, rendering them useful for research. Herein, considerable myelin loss is shown in the optic nerves of cuprizone-treated demyelinating mice. Immuno-fluorescence confocal analysis of the expression and distribution of voltage-activated K⁺ channels (K(V)1.1 and 1.2 α subunits) revealed their spread from typical juxta-paranodal (JXP) sites to nodes in demyelinated axons, albeit with a disproportionate increase in the level of K(V)1.1 subunit. Functionally, in contrast to monophasic compound action potentials (CAPs) recorded in controls, responses derived from optic nerves of cuprizone-treated mice displayed initial synchronous waveform followed by a dispersed component. Partial restoration of CAPs by broad spectrum (4-aminopyridine) or K(V)1.1-subunit selective (dendrotoxin K) blockers of K⁺ currents suggest enhanced K(V)1.1-mediated conductance in the demyelinated optic nerve. Biophysical profiling of K⁺ currents mediated by recombinant channels comprised of different K(V)1.1 and 1.2 stoichiometries revealed that the enrichment of K(V)1 channels K(V)1.1 subunit endows a decrease in the voltage threshold and accelerates the activation kinetics. Together with the morphometric data, these findings provide important clues to a molecular basis for temporal dispersion of CAPs and reduced excitability of demyelinated optic nerves, which could be of potential relevance to the patho-physiology of MS and related disorders.

Neurospheres derived from human embryoid bodies treated with retinoic Acid show an increase in nestin and ngn2 expression that correlates with the proportion of tyrosine hydroxylase-positive cells.

In the central nervous system (CNS), generation of phenotypic diversity within the neuronal lineage is precisely regulated in a spatial and temporal fashion. Neural basic helix-loop-helix (bHLH) transcription factors are cell intrinsic factors that control commitment to neuronal lineage and play an important role in neuronal cell type specification. The ability to differentiate human embryonic stem (hES) cells into neurons provides a good model system to address human neuronal specification. Previous studies have shown neurogenin-2 (Ngn2) to be involved in the development of mesencephalic dopaminergic neurons. Toward the goal of correlating neuronal phenotype with early gene expression pattern, we have characterized the expression of Ngn2 during hES cell differentiation. Our results show that treatment of embryoid bodies (EBs) with retinoic acid (RA) leads to the greatest proportion of tyrosine hydroxylase (TH)-positive cells followed by vasoactive intestinal peptide (VIP)-treated EBs as compared to untreated EBs. This increase in the proportion of TH-positive neurons was correlated with the unique morphology of RA-treated aggregates and the spatial delocalization of the expression of Ngn2 within the EB. Neurospheres derived from RA-treated EBs contained many nestin-positive cells within regions that expressed Ngn2. We show that the extent of nestin-positive cells that arise from the region of Ngn2 expression is correlated with the appearance of TH-positive neurons. Our results show for the first time the expression of Ngn2 during the differentiation of hES cells.

CMV promotor activity during ES cell differentiation: potential insight into embryonic stem cell differentiation.

The activity of the P(CMV IE) promoter was studied during the differentiation of ES cells into neurons. In order to do this, stable embryonic stem (ES) cell lines that express enhanced green fluorescent protein (EGFP) under the control of P(CMV IE) were created and these ES cells were differentiated by aggregation of cells in the presence of retinoic acid (RA). Based on our observations that the activity of P(CMV IE) was highest in undifferentiated cells, and that cell-cell interaction and addition of RA that lead to enhanced cell proliferation also increased expression from P(CMV IE), we hypothesized that the activity of P(CMV IE) was positively regulated in cycling cells. However, when analysis was done at the single cell level it was found that BrdU label and EGFP expression were not correlated. EGFP expression was found to be down-regulated in many cells that were BrdU positive and conversely there were significant numbers of BrdU negative cells that were EGFP positive. Further, P(CMV IE) activity was not observed in cells that were nestin positive or in differentiated neurons, but P(CMV IE) was active in cells with a fibroblast-like morphology. Finally, several proteins present in undifferentiated ES cells were found to bind to regulatory regions of P(CMV IE). These were absent when cells were aggregated in the presence of RA. The above results have implications for expression of transgenes in ES cells as well as providing new insight into the mechanism of lineage restriction.