We developed a method for the efficient generation of functional dopaminergic (DA) neurons from human embryonic stem cells (hESCs) on a large scale. The most unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the hESC-derived neural precursors. These SNMs provide several advantages: (i) they can be passaged for a long time without losing their differentiation capability into DA neurons; (ii) they can be coaxed into DA neurons at much higher efficiency than that from previous reports (86% tyrosine hydroxylase-positive neurons/total neurons); (iii) the induction of DA neurons from SNMs only takes 14 days; and (iv) no feeder cells are required during differentiation. These advantages allowed us to obtain a large number of DA neurons within a short time period and minimized potential contamination of unwanted cells or pathogens coming from the feeder layer. The highly efficient differentiation may not only enhance the efficacy of the cell therapy but also reduce the potential tumor formation from the undifferentiated residual hESCs. In line with this effect, we have never observed any tumor formation from the transplanted animals used in our study. When grafted into a parkinsonian rat model, the hESC-derived DA neurons elicited clear behavioral recovery in three behavioral tests. In summary, our study paves the way for the large-scale generation of purer and functional DA neurons for future clinical applications.is a neurodegenerative disorder characterized by progressive and selective loss of dopaminergic (DA) neurons in the midbrain substantia nigra (1). Currently, the prevailing strategy for the treatment of PD is pharmacological. However, pharmacological treatment with L-DOPA works initially, but over time, the effectiveness of L-DOPA wanes and side effects develop (2). An alternative approach may be the transplantation of DA-synthesizing cells. One source of DA-synthesizing cells is embryonic stem cells (ESCs). ESCs are pluripotent and capable of self-renewal (3-5). For the purpose of applying the ESCs to PD, many researchers have tried to develop protocols by which ESCs from some species can differentiate into DA neuronal phenotypes (6-11). Although some progress has been made in the generation of DA neurons from human ESCs (hESCs) (12-22), there are still many technical improvements to be made before the application of hESCs to treat PD. Examples include increasing the purity of DA neurons, supplying a sufficient quantity of DA neurons for clinical applications, decreasing tumor formation after transplantation, and clearly demonstrating the functionality of hESC-derived DA neurons in a parkinsonian animal model.Here, we introduce a method that allows us to differentiate hESCs into functional tyrosine hydroxylase-positive (TH ϩ ) neurons up to near 86% of the total hESC-derived neurons, which is the highest purity ever reported. Achieving high efficiency of DA neuronal derivation is an important issue in cell therapy, because it would not only increase the effica...

Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is essential for compacted heterochromatin structure and associated gene silencing. Its chromo shadow domain (CSD) is well-known for binding to peptides that contain a PXVXL motif. Heterochromatin protein 2 (HP2) is a nonhistone chromosomal protein that associates with HP1a in the pericentric heterochromatin, telomeres and the fourth chromosome. Using NMR spectroscopy, fluorescence polarization and site-directed mutagenesis, we identified an LCVKI motif in HP2 that binds to the HP1a CSD. The binding affinity of the HP2 fragment is approximately two orders of magnitude higher than that of peptides from PIWI (with a PRVKV motif), AF10 (with a PLVVL motif), or CG15356 (with LYPLL and LSIVA motifs). To delineate differential interactions of the HP1a CSD, we characterized its structure, backbone dynamics and dimerization constant. We find that the dimerization constant is bracketed by the affinities of HP2 and PIWI, which dock to the same HP1a homodimer surface. This suggests that HP2, but not PIWI, interaction can drive homodimerization of HP1a. Interestingly, the integrity of the disordered C-terminal extension (CTE) of HP1a is essential for discriminatory binding, whereas swapping the PXVXL motifs does not confer specificity. Serine phosphorylation at the peptide binding surface of the CSD is thought to regulate heterochromatin assembly. Glutamic acid substitution at these sites destabilizes HP1a dimers, but improves the interaction with both binding partners. Our studies underscore the importance of CSD dimerization and cooperation with the CTE in forming distinct complexes of HP1a.

MSL3 resides in the MSL (male-specific-lethal) complex that upregulates transcription by spreading the H4K16 acetyl-mark. We discovered a DNA-dependent interaction of MSL3 chromodomain with the histone H4K20 monomethyl-mark. Structure of a ternary complex shows DNA minor groove accommodates the histone H4 tail, and monomethyllysine inserts in a four-residue aromatic cage in MSL3. Histone H4K16 acetyl-mark antagonizes MSL3 binding, suggesting MSL function is regulated by a combination of post-translational modifications.

Previously, we found that amyloid b-protein (Ab)1-42 exhibits neurotoxicity, while Ab1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Ab1-40 against Ab1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Ab1-42 at concentrations higher than 2 lM, which was prevented by concurrent treatment with Ab1-40 in a dosedependent manner. However, metal chelators did not prevent Ab1-42-induced neuronal death. Circular dichroism spectroscopy showed that Ab1-40 inhibited the b-sheet transformation of Ab1-42. Thioflavin-T assay and electron microscopy analysis revealed that Ab1-40 inhibited the fibril formation of Ab1-42. In contrast, Ab1-16, Ab25-35, and Ab40-1 did not inhibit the fibril formation of Ab1-42 nor prevent Ab1-42-induced neuronal death. Ab1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Ab1-40. These results indicate that Ab1-40 protects neurons from Ab1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the b-sheet transformation and fibril formation of Ab1-42. Our data suggest a mechanism by which elevated Ab1-42/Ab1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.