Human-induced pluripotent stem cells as a model for studying sporadic Alzheimer's disease.

The discovery of induced pluripotent stem cell (iPSC) technology has the potential to accelerate scientific research for Alzheimer's disease (AD). iPSCs are therefore increasingly considered for AD modeling and drug development. Nevertheless, most of the work conducted so far has mainly focused on iPSC models from patients with familial AD (fAD), while actually sporadic AD (sAD) is more prevalent and represents over 90% of the AD cases in the population. The development of more sAD models is therefore key for studying this multifactorial disorder. In fact, probing the unique genomes of sAD patients and their interaction with AD-associated environmental factors could contribute to a better understanding of this disease. However, initial iPSC-based models for sAD have shown a high degree of variability and inconsistencies in terms of AD hallmarks. In this review, we provide an overview of the studies that have been conducted for sAD so far. In addition, we critically assess important sources of variability related to the model in addition to those that might be explained by the heterogeneous nature of sAD. These considerations might aid in developing more consistent iPSC models of sAD, which could help in developing a better understanding of the molecular mechanisms underlying the disease.

Valproic acid promotes mitochondrial dysfunction in primary human hepatocytes in vitro; impact of C/EBPα-controlled gene expression.

Valproic acid (VPA) is a frequently prescribed anti-epileptic drug which is known to cause liver toxicity and steatosis through mitochondrial dysfunction. Nevertheless the mechanisms underlying these adverse effects are incompletely understood. In this study, we determined the effect of relatively short (3 h) or prolonged (72 h) exposure to VPA on mitochondrial function in primary human hepatocytes (PHHs). While 3 h VPA exposure did not affect oxygen consumption rates (OCRs) in PHHs, prolonged exposure (24-72 h) significantly reduced basal and maximal OCRs. Given that in particular prolonged VPA exposure is required to cause mitochondrial dysfunction, we investigated gene expression data after VPA exposure for 24, 48, 72 h and 72 h VPA followed by a 72 h washout period. We were able to reduce the comprehensive gene expression changes into a more comprehensible set of 18 TFs that were predicted to be persistently activated after 72 h of VPA exposure. Lentiviral knock-down of one of the candidate TFs, C/EBPα, partly rescued VPA-induced mitochondrial dysfunction. Furthermore, RNA-Seq analysis of shC/EBPα and shGFP control PHHs identified 24 genuine C/EBPα target genes that are regulated in response to prolonged VPA exposure in PHHs. Altogether this provides new insights on the involvement of C/EBPα in driving VPA-induced mitochondrial dysfunction in human liver cells. This hub gene, with its downstream regulators involved in this deregulation, thus represent potential new biomarkers for VPA-induced mitochondrial dysfunction.

Inferring transcription factor activity from microarray data reveals novel targets for toxicological investigations.

Transcription factors (TFs) are important modulators of the inducible portion of the transcriptome, and therefore relevant in the context of exposure to exogenous compounds. Current approaches to predict the activity of TFs in biological systems are usually restricted to a few entities at a time due to low-throughput techniques targeting a limited fraction of annotated human TFs. Therefore, high-throughput alternatives may help to identify new targets of mechanistic and predictive value in toxicological investigations. In this study, we inferred the activity multiple TFs using publicly available microarray data from primary human hepatocytes exposed to hundreds of chemicals and evaluated these molecular profiles using multiple correspondence analysis. Our results demonstrate that the lowest dose and latest exposure time (24h) in a subset of chemicals generates a signature indicative of carcinogenicity possibly due to DNA-damaging properties. Furthermore, profiles from the earliest exposure time (2h) and highest dose creates clusters of chemicals implicated in the development of diverse forms of drug-induced liver injury (DILI). Both approaches yielded a number of TFs with similar activity across groups of chemicals, including TFs known in toxicological responses such as AhR, NFE2L2 (Nrf2), NF-κB and PPARG. FOXM1, IRF1 and E2F4 were some of the TFs identified that may be relevant in genotoxic carcinogenesis. SMADs (SMAD1, SMAD2, SMAD5) and KLF5 were identified as some of potentially new TFs whose inferred activities were linked to acute and progressive outcomes in DILI. In conclusion this study offers a novel mechanistic approach targeting TF activity during chemical exposure.

Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains.

Ligands specific for B7.1 (CD80) and B7.2 (CD86) have applications in disease indications that require inhibition of T-cell activity. As we observed significant sequence and structural similarity between the B7-binding ligand, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and antibody variable light chain domains (VLs), we have explored the possibilities of making novel B7 binding molecules based on single VL domains. We first describe the "rational" design and construction of a VL/CTLA-4 hybrid molecule in which we have grafted both the CDR1 and CDR3-like loops of CTLA-4 onto a single VL light chain, at sites determined by sequence and structure-based alignment. This molecule was secreted as a soluble product from Escherichia coli, but did not show any binding to B7.1 and B7.2. In a second approach we constructed a VL library in which human VL genes derived from B-cells were spiked with the CDR3-like loop of CTLA-4 and further diversified by DNA shuffling. This library was displayed on phage, and after selection gave B7.1 binding ligands which competed with CTLA-4. In order to evaluate the possible general utility of VL domains as binding ligands, we have constructed a non-biased VL library. From this DNA-shuffled human VL library we have selected single VL domains specific for B7.1, B7.2 or human IgG. Two B7.1-specific VL ligands and one B7.2-specific VL ligand showed competition with CTLA-4. One candidate VL domain-specific for B7.1 was affinity matured by simultaneous randomisation of all CDR loops using DNA shuffling with degenerate CDR-spiking oligonucleotides. From this library, a single VL domain with affinity of 191 nM for B7.1 was obtained, which also showed binding to B7.1 in situ. This VL had mutations in CDR1 and CDR3, indicating that antigen recognition for this single VL is most likely mediated by the same regions as in the VL domain of whole antibodies. The B7.1 and B7.2-specific VL domains described in this study may form the basis of a new family of immunomodulatory recombinant molecules. Furthermore, our studies suggest that it is feasible to create specific single VL domains to diverse targets as is the case for single VH domains.