Induction of neural differentiation by the transcription factor neuroD2.

Pro-neural basic helix loop helix (bHLH) transcription factors are involved in many aspects of normal neuronal development, and over-expression of genes for several of these factors has been shown to induce aspects of neuronal differentiation in cell lines and stem cells. Here we show that over-expression of NeuroD2 (ND2), Neurogenin1 and 2 leads to morphological differentiation of N18-RE-105 neuroblastoma cells and increased expression of synaptic proteins. Particularly ND2 induced neurite formation and increases in the expression of synaptic proteins such as synaptotagmin, that is not expressed normally in this cell type, as well as the redistribution of another synaptic protein, SNAP25, to a cell membrane location. Infection of human neural progenitor cells using adeno associated viral (AAV) vectors also promoted neuronal differentiation. Over-expressing cells demonstrated a significant increase in the neuron specific form of tubulin as well as increased expression of synaptotagmin. Genetic modification of neural progenitor cell with bHLH factors such as ND2 may be a viable strategy to enhance differentiation of these cells into replacement neurons for human disease.

Induction of tyrosine hydroxylase expression by the transcription factor Pitx3.

Transcription factors are fate determining regulatory factors in dopaminergic neuronal development and differentiation. Among them, Nurr1 is the most extensively studied, but the importance of Pitx3 has recently been appreciated. Over-expression of both factors has been utilized to enhance the dopaminergic differentiation of stem cells for transplantation into models of Parkinson's disease. Previous studies however have seen conflicting results regarding the induction of tyrosine hydroxylase expression and dopaminergic differentiation induced by over-expression of Pitx3. Here we show that over-expression of Pitx3 and Nurr1 induced endogenous tyrosine hydroxylase expression as well as a tyrosine hydroxylase promoter-reporter construct in a human non-neuronal and mouse embryonic stem cell lines. Combined simultaneous expression of Nurr1 and Pitx3 however did not lead to enhancement of tyrosine hydroxylase expression over that of either factor alone in either of the cell lines or with either method. These results suggest that other regulatory elements may also be involved in regulation of tyrosine hydroxylase expression. There was also a lack of a correlation between the expression levels of tyrosine hydroxylase with that of the transcription factor constructs. To yield a robust dopaminergic differentiation a combinatorial or successive treatment with different transcription factors may be more effective.

An in vitro model of inflammatory neurodegeneration and its neuroprotection.

Inflammation has been implicated in a variety of acute and chronic neurodegenerative diseases in which the inflammatory processes are considered not only to result from neurodegenerative effects, but also to contribute to these effects. To investigate the primary effect of inflammation on neuronal survival, a co-culture system of neuronal cells (differentiated SH SY5Y human neuroblastoma cells or primary cortical/striatal neurons) and monocytic cells (THP-1) in direct cell-cell contact was set up. After 5 days, THP-1 activation by lipopolysaccharide and phorbol 12-myristate 13-acetate resulted in a significant increase of neuronal cell death compared to co-culture without activation. In neuroprotection studies using this model, ascorbic acid and EDTA demonstrated a highly significant reduction in activated THP-1 induced cell death. Glutathione and NBQX, but not the protease inhibitor, PMSF, and catalase, also significantly reduced this inflammatory neurotoxicity. Indomethacin was protective of the primary cultured neurons but not the SH SY5Y cells. This co-culture of neuronal cells and activated THP-1 provides a useful model for the study of inflammatory mechanisms resulting in neuronal cell death.