Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.Initiative Joint Undertaking under grant agreement no. 115439, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies' in kind contribution. A.H., S.C., and M.Z.C. were also funded by the NIHR (Oxford BRC). K.M. and A.B. were also supported by the NIHR GOSH BRC

Link-Rot in Web-Sourced Multimedia Datasets

The Web is increasingly used as a source for content of datasets of various types, especially multimedia content. These datasets are then often distributed as a collection of URLs, pointing to the original sources of the elements. As these sources go offline over time, the datasets experience decay in the form of link-rot. In this paper, we analyze 24 Web-sourced datasets with a combined total of over 270 million URLs and find that over 20% of the content is no longer available. We discuss the adverse effects of this decay on the reproducibility of work based on such data and make some recommendations on how they could be mediated in the future

N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells—implications for axon ensheathment at myelination

The molecular mechanisms used by oligodendrocyte precursor cells (OPCs), oligodendrocytes (OLs), and Schwann cells (SCs) to advance processes for motility in the developing nervous system and to ensheath axons at myelination are currently not well defined. Here we demonstrate that OPCs, OLs, and SCs express the major proteins involved in actin polymerization-driven protrusion; these key proteins including F-actin, the Arp2/3 complex, neural-Wiskott Aldrich Syndrome protein (N-WASP) and WAVE proteins, and the RhoGTPases Rac and Cdc42 are present at the leading edges of processes being extended by OPCs, OLs, and SCs. We reveal by real-time PCR that OLs and SCs have different dominant WAVE isoforms. Inhibition of the WASP/WAVE protein, N-WASP, with wiskostatin that prevents activation of the Arp2/3 complex, blocks process extension by OPCs and SCs. Inhibition of N-WASP also causes OPC and SC process retraction, which is preceded by retraction of filopodia. This implicates filopodia in OPC and SC process stability and also of N-WASP in OPC and SC process dynamics. We also demonstrate that p34 (a component of the Arp2/3 complex), WASP/WAVE proteins, actin, α-tubulin, Rac, Cdc42, vinculin, and focal adhesion kinase are detected in water-shocked myelin purified from brain. Inhibition of N-WASP with wiskostatin decreases the number of axons undergoing initial ensheathment in intact optic nerve samples and reduces the Po content of dorsal root ganglia:SC co-cultures. Our findings indicate that OPCs, OLs, and SCs extend processes using actin polymerization-driven protrusion dependent on N-WASP. We hypothesize that inner mesaxons of OLs and SCs use the same mechanism to ensheath axons at myelination

Isoform- and cell-state-specific lipidation of ApoE in astrocytes

Apolipoprotein E transports lipids and couples metabolism between astrocytes and neurons. The E4 variant (APOE4) affects these functions and represents a genetic predisposition for Alzheimer's disease, but the molecular mechanisms remain elusive. We show that ApoE produces different types of lipoproteins via distinct lipidation pathways. ApoE forms high-density lipoprotein (HDL)-like, cholesterol-rich particles via the ATP-binding cassette transporter 1 (ABCA1), a mechanism largely unaffected by ApoE polymorphism. Alternatively, ectopic accumulation of fat in astrocytes, a stress-associated condition, redirects ApoE toward the assembly and secretion of triacylglycerol-rich lipoproteins, a process boosted by the APOE4 variant. We demonstrate in vitro that ApoE can detect triacylglycerol in membranes and spontaneously assemble lipoprotein particles (10–20 nm) rich in unsaturated triacylglycerol, and that APOE4 has remarkable properties behaving as a strong triacylglycerol binder. We propose that fatty APOE4 astrocytes have reduced ability to clear toxic fatty acids from the extracellular milieu, because APOE4 reroutes them back to secretion.ISSN:2666-3864ISSN:2211-124

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.Initiative Joint Undertaking under grant agreement no. 115439, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies' in kind contribution. A.H., S.C., and M.Z.C. were also funded by the NIHR (Oxford BRC). K.M. and A.B. were also supported by the NIHR GOSH BRC

The Methylazoxymethanol Acetate (MAM-E17) Rat Model: Molecular and Functional Effects in the Hippocampus

Administration of the DNA-alkylating agent methylazoxymethanol acetate (MAM) on embryonic day 17 (E17) produces behavioral and anatomical brain abnormalities, which model some aspects of schizophrenia. This has lead to the premise that MAM rats are a neurodevelopmental model for schizophrenia. However, the underlying molecular pathways affected in this model have not been elucidated. In this study, we investigated the molecular phenotype of adult MAM rats by focusing on the frontal cortex and hippocampal areas, as these are known to be affected in schizophrenia. Proteomic and metabonomic analyses showed that the MAM treatment on E17 resulted primarily in deficits in hippocampal glutamatergic neurotransmission, as seen in some schizophrenia patients. Most importantly, these results were consistent with our finding of functional deficits in glutamatergic neurotransmission, as identified using electrophysiological recordings. Thus, this study provides the first molecular evidence, combined with functional validation, that the MAM-E17 rat model reproduces hippocampal deficits relevant to the pathology of schizophrenia