Generation of a set of induced pluripotent stem cell lines from two Alzheimer disease patients carrying APOE4 (MLUi007-J; MLUi008-A) and healthy old donors carrying APOE3 (MLUi009-A; MLUi010-B) to study APOE in aging and disease.

Late-onset Alzheimer disease (LOAD) is the most frequent neurodegenerative disease, and the APOE ε4 allele is the most prominent risk factor for LOAD. Four human induced pluripotent stem cell (iPSC) lines MLUi007-J, MLUi008-B, MLUi009-A, and MLUi010-B were generated from LOAD patients and healthy matched donors by reprogramming of B-lymphoblastoid cells (B-LCLs) with episomal plasmids. The application of B-LCLs holds a great promise to model LOAD and other diseases because they can easily be generated from primary peripheral blood mononuclear cells (PBMCs) by infection with the Epstein-Barr virus (EBV).

Targeting autophagy in disease: established and new strategies.

Macroautophagy/autophagy is an evolutionarily conserved pathway responsible for clearing cytosolic aggregated proteins, damaged organelles or invading microorganisms. Dysfunctional autophagy leads to pathological accumulation of the cargo, which has been linked to a range of human diseases, including neurodegenerative diseases, infectious and autoimmune diseases and various forms of cancer. Cumulative work in animal models, application of genetic tools and pharmacologically active compounds, has suggested the potential therapeutic value of autophagy modulation in disease, as diverse as Huntington, Salmonella infection, or pancreatic cancer. Autophagy activation versus inhibition strategies are being explored, while the role of autophagy in pathophysiology is being studied in parallel. However, the progress of preclinical and clinical development of autophagy modulators has been greatly hampered by the paucity of selective pharmacological agents and biomarkers to dissect their precise impact on various forms of autophagy and cellular responses. Here, we summarize established and new strategies in autophagy-related drug discovery and indicate a path toward establishing a more efficient discovery of autophagy-selective pharmacological agents. With this knowledge at hand, modern concepts for therapeutic exploitation of autophagy might become more plausible.Abbreviations: ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related gene; AUTAC: autophagy-targeting chimera; CNS: central nervous system; CQ: chloroquine; GABARAP: gamma-aminobutyric acid type A receptor-associated protein; HCQ: hydroxychloroquine; LYTAC: lysosome targeting chimera; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NDD: neurodegenerative disease; PDAC: pancreatic ductal adenocarcinoma; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PROTAC: proteolysis-targeting chimera; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.

An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds.

The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting. Graphical abstract •The embryonic stem cell test to predict teratogenicity was made automation-compatible. •Several key improvements to the assay procedure have been introduced to increase performance. •The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.

T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder.

While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD) is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR) repertoire in MDD. For this cross-sectional case-control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (n = 20), who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (n = 20). T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4+ T cell compartment characterized by a higher frequency of CD4+CD25highCD127low/- cells and higher FOXP3 mRNA expression in purified CD4+ T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vß repertoire analysis indicated a less diverse CD4+ T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR ß chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder.

Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes.

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5' or 3' pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.

A screening strategy for the discovery of drugs that reduce C/EBPß-LIP translation with potential calorie restriction mimetic properties.

An important part of the beneficial effects of calorie restriction (CR) on healthspan and lifespan is mediated through regulation of protein synthesis that is under control of the mechanistic target of rapamycin complex 1 (mTORC1). As one of its activities, mTORC1 stimulates translation into the metabolic transcription factor CCAAT/Enhancer Binding Protein ß (C/EBPß) isoform Liver-specific Inhibitory Protein (LIP). Regulation of LIP expression strictly depends on a translation re-initiation event that requires a conserved cis-regulatory upstream open reading frame (uORF) in the C/EBPß-mRNA. We showed before that suppression of LIP in mice, reflecting reduced mTORC1-signaling at the C/EBPß level, results in CR-type of metabolic improvements. Hence, we aim to find possibilities to pharmacologically down-regulate LIP in order to induce CR-mimetic effects. We engineered a luciferase-based cellular reporter system that acts as a surrogate for C/EBPß-mRNA translation, emulating uORF-dependent C/EBPß-LIP expression under different translational conditions. By using the reporter system in a high-throughput screening (HTS) strategy we identified drugs that reduce LIP. The drug Adefovir Dipivoxil passed all counter assays and increases fatty acid ß-oxidation in a hepatoma cell line in a LIP-dependent manner. Therefore, these drugs that suppress translation into LIP potentially exhibit CR-mimetic properties.

Effects of exercise on Irisin, BDNF and IL-6 serum levels in patients with progressive multiple sclerosis.

BACKGROUND: Clinical studies have suggested beneficial effects of exercise on cognitive function in ageing adults and neurodegenerative diseases such as dementia. Recent work indicates the same for progressive multiple sclerosis (MS), an inflammatory and degenerative disease of the central nervous system (CNS). The biological pathways associated with these effects are however not well understood. OBJECTIVE: In this randomized controlled study, we explored serum levels of the myokine Irisin, the neurotrophin brain-derived neurotrophic factor (BDNF) and Interleukin-6 (IL-6) during acute endurance exercise and over the course of a 9-weeks endurance exercise training period in n=42 patients with progressive MS. RESULTS: We detected a significant increase of BDNF levels in progressive MS patients after 30min of bicycling (p<0.001). However, there were no significant changes for baseline levels after 22 sessions of training. No significant effects of acute or prolonged exercise could be found for Irisin or Interleukin-6. CONCLUSION: Our results indicate that BDNF is strongly induced during acute exercise even in patients with progressive MS and advanced physical disability. Long-term effects of exercise programs on biological parameters (Irisin, BDNF, IL-6) were much less pronounced. Given the hypothesis-driven selection of a limited set of biological markers in this pilot study, future studies should use unbiased approaches in larger samples to obtain a comprehensive picture of the networks involved in exercise effects on neurological diseases.

A multiple sclerosis-associated variant of CBLB links genetic risk with type I IFN function.

Multiple sclerosis (MS) is an autoimmune disease of the CNS, and autoreactive CD4(+) T cells are considered important for its pathogenesis. The etiology of MS involves a complex genetic trait and environmental triggers that include viral infections, particularly the EBV. Among the risk alleles that have repeatedly been identified by genome-wide association studies, three are located near the Casitas B-lineage lymphoma proto-oncogene b gene (CBLB). The CBLB protein (CBL-B) is a key regulator of peripheral immune tolerance by limiting T cell activation and expansion and hence T cell-mediated autoimmunity through its ubiquitin E3-ligase activity. In this study, we show that CBL-B expression is reduced in CD4(+) T cells from relapsing-remitting MS (RR-MS) patients during relapse. The MS risk-related single nucleotide polymorphism of CBLB rs12487066 is associated with diminished CBL-B expression levels and alters the effects of type I IFNs on human CD4(+) T cell proliferation. Mechanistically, the CBLB rs12487066 risk allele mediates increased binding of the transcription factor C/EBPß and reduced CBL-B expression in human CD4(+) T cells. Our data suggest a role of the CBLB rs12487066 variant in the interactions of a genetic risk factor and IFN function during viral infections in MS.

Functional role of the interaction between polysialic acid and myristoylated alanine-rich C kinase substrate at the plasma membrane.

Polysialic acid (PSA) is a homopolymeric glycan that plays crucial roles in the developing and adult nervous system. So far only a few PSA-binding proteins have been identified. Here, we identify myristoylated alanine-rich C kinase substrate (MARCKS) as novel PSA binding partner. Binding assays showed a direct interaction between PSA and a peptide comprising the effector domain of MARCKS (MARCKS-ED). Co-immunoprecipitation of PSA-carrying neural cell adhesion molecule (PSA-NCAM) with MARCKS and co-immunostaining of MARCKS and PSA at the cell membrane of hippocampal neurons confirm the interaction between PSA and MARCKS. Co-localization and an intimate interaction of PSA and MARCKS at the cell surface was seen by confocal microscopy and fluorescence resonance energy transfer (FRET) analysis after the addition of fluorescently labeled PSA or PSA-NCAM to live CHO cells or hippocampal neurons expressing MARCKS as a fusion protein with green fluorescent protein (GFP). Cross-linking experiments showed that extracellularly applied PSA or PSA-NCAM and intracellularly expressed MARCKS-GFP are in close contact, suggesting that PSA and MARCKS interact with each other at the plasma membrane from opposite sides. Insertion of PSA and MARCKS-ED peptide into lipid bilayers from opposite sides alters the electric properties of the bilayer confirming the notion that PSA and the effector domain of MARCKS interact at and/or within the plane of the membrane. The MARCKS-ED peptide abolished PSA-induced enhancement of neurite outgrowth from cultured hippocampal neurons indicating an important functional role for the interaction between MARCKS and PSA in the developing and adult nervous system.

A differential proteome screening system for post-translational modification-dependent transcription factor interactions.

Post-translational modifications (PTMs) of transcription factors alter interactions with co-regulators and epigenetic modifiers. For example, members of the C/EBP transcription factor family are extensively methylated on arginine and lysine residues in short, conserved, modular domains, implying modification-dependent cofactor docking. Here we describe array peptide screening (APS), a systematic and differential approach to detect PTM-dependent interactions in the human proteome using chemically synthesized, biotinylated peptides coupled to fluorophore-labeled streptavidin. Peptides with and without a modified residue are applied in parallel to bacterial expression libraries in an arrayed format. Interactions are detected and quantified by laser scanning to reveal proteins that differentially bind to nonmodified or modified peptides. We have previously used this method to investigate the effect of arginine methylation of C/EBPß peptides. The method enables determination of PTM-dependent transcription factor interactions, quantification of relative binding affinities and rapid protein classification, all independently of the transactivation potential of peptides or cellular abundance of interactors. The protocol provides a cost-effective alternative to mass spectrometric approaches and takes 3-4 d to complete.

Crosstalk between phosphorylation and multi-site arginine/lysine methylation in C/EBPs.

C/EBPs are implied in an amazing number of cellular functions: C/EBPs regulate tissue and cell type specific gene expression, proliferation, and differentiation control. C/EBPs assist in energy metabolism, female reproduction, innate immunity, inflammation, senescence, and the development of neoplasms. How can C/EBPs fulfill so many functions? Here we discuss that C/EBPs are extensively modified by methylation of arginine and lysine side chains and that regulated methylation profoundly affects the activity of C/EBPs.

Crosstalk between C/EBPbeta phosphorylation, arginine methylation, and SWI/SNF/Mediator implies an indexing transcription factor code.

Cellular signalling cascades regulate the activity of transcription factors that convert extracellular information into gene regulation. C/EBPbeta is a ras/MAPkinase signal-sensitive transcription factor that regulates genes involved in metabolism, proliferation, differentiation, immunity, senescence, and tumourigenesis. The protein arginine methyltransferase 4 PRMT4/CARM1 interacts with C/EBPbeta and dimethylates a conserved arginine residue (R3) in the C/EBPbeta N-terminal transactivation domain, as identified by mass spectrometry of cell-derived C/EBPbeta. Phosphorylation of the C/EBPbeta regulatory domain by ras/MAPkinase signalling abrogates the interaction between C/EBPbeta and PRMT4/CARM1. Differential proteomic screening, protein interaction studies, and mutational analysis revealed that methylation of R3 constraines interaction with SWI/SNF and Mediator complexes. Mutation of the R3 methylation site alters endogenous myeloid gene expression and adipogenic differentiation. Thus, phosphorylation of the transcription factor C/EBPbeta couples ras signalling to arginine methylation and regulates the interaction of C/EBPbeta with epigenetic gene regulatory protein complexes during cell differentiation.

Targets of the Tal1 transcription factor in erythrocytes: E2 ubiquitin conjugase regulation by Tal1.

The Tal1 transcription factor is essential for the development of the hematopoietic system and plays a role during definitive erythropoiesis in the adult. Despite the importance of Tal1 in erythropoiesis, only a small number of erythroid differentiation target genes are known. A chromatin precipitation and cloning approach was established to uncover novel Tal1 target genes in erythropoiesis. The BirA tag/BirA ligase biotinylation system in combination with streptavidin chromatin precipitation (Strep-CP) was used to co-precipitate genomic DNA bound to Tal1. Tal1 was found to bind in the vicinity of 31 genes including the E2-ubiquitin conjugase UBE2H gene. Binding of Tal1 to UBE2H was confirmed by chromatin immunoprecipitation. UBE2H expression is increased during erythroid differentiation of hCD34(+) cells. Tal1 expression activated UBE2H expression, whereas Tal1 knock-down reduced UBE2H expression and ubiquitin transfer activity. This study identifies parts of the ubiquitinylation machinery as a cellular target downstream of the transcription factor Tal1 and provides novel insights into Tal1-regulated erythropoiesis.