Dentate granule cell (DGC) neurogenesis persists throughout life in the mammalian hippocampal dentate gyrus and increases after epileptogenic insults. The DGC layer in human and experimental mesial temporal lobe epilepsy (mTLE) often shows abnormal dispersion and the appearance of hilar-ectopic DGCs. In the pilocarpine mTLE model, hilar-ectopic DGCs arise as a result of an aberrant chain migration of neural progenitors. Reelin is a secreted migration guidance cue that persists in the adult rodent and human hippocampus. We tested the hypothesis that loss of Reelin in the epileptic dentate gyrus leads to aberrant chain migration of DGC precursors. We found that interneuron subsets typically lost in human and experimental mTLE express Reelin, and DGC progenitors express the downstream Reelin signaling molecule Disabled 1 (Dab1). Prolonged seizures decreased Reelin immunoreactivity in the adult rat dentate gyrus and increased Dab1 expression in hilar-ectopic neuroblasts. Exogenous Reelin increased detachment of chain-migrating neuroblasts in dentate gyrus explants, and blockade of Reelin signaling increased chain migration. These findings suggest that Reelin modulates DGC progenitor migration to maintain normal DGC integration in the neonatal and adult mammalian dentate gyrus. Loss of Reelin expression in the epileptic adult hippocampus, moreover, likely contributes to ectopic chain migration and aberrant integration of newborn DGCs.

The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.

The basic helix-loop-helix transcription factor Ascl1 plays a critical role in the intrinsic genetic program responsible for neuronal differentiation. Here, we describe a novel model system of P19 embryonic carcinoma cells with doxycycline-inducible expression of Ascl1. Microarray hybridization and real-time PCR showed that these cells demonstrated increased expression of many neuronal proteins in a time-and concentration-dependent manner. Interestingly, the gene encoding the cell cycle regulator Gadd45γ was increased earliest and to the greatest extent following Ascl1 induction. Here, we provide the first evidence identifying Gadd45γ as a direct transcriptional target of Ascl1. Transactivation and chromatin immunoprecipitation assays identified two E-box consensus sites within the Gadd45γ promoter necessary for Ascl1 regulation, and demonstrated that Ascl1 is bound to this region within the Gadd45γ promoter. Furthermore, we found that overexpression of Gadd45γ itself is sufficient to initiate some aspects of neuronal differentiation independent of Ascl1.

As members of the proneural basic-helix-loop-helix (bHLH) family of transcription factors, Ascl1 and Neurog2 direct the differentiation of specific populations of neurons at various times and locations within the developing nervous system. In order to characterize the mechanisms employed by these two bHLH factors, we generated stable, doxycycline-inducible lines of P19 embryonic carcinoma cells that express comparable levels of Ascl1 and Neurog2. Upon induction, both Ascl1 and Neurog2 directed morphological and immunocytochemical changes consistent with initiation of neuronal differentiation. Comparison of Ascl1- and Neurog2-regulated genes by microarray analyses showed both shared and distinct transcriptional changes for each bHLH protein. In both Ascl1- and Neurog2-differentiating cells, repression of Oct4 mRNA levels was accompanied by increased Oct4 promoter methylation. However, DNA demethylation was not detected for genes induced by either bHLH protein. Neurog2-induced genes included glutamatergic marker genes while Ascl1-induced genes included GABAergic marker genes. The Neurog2-specific induction of a gene encoding a protein phosphatase inhibitor, Ppp1r14a, was dependent on distinct, canonical E-box sequences within the Ppp1r14a promoter and the nucleotide sequences within these E-boxes were partially responsible for Neurog2-specific regulation. Our results illustrate multiple novel mechanisms by which Ascl1 and Neurog2 regulate gene repression during neuronal differentiation in P19 cells.