Proteomics of mouse brain endothelium uncovers dysregulation of vesicular transport pathways during aging.

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging.

Reproducible and scalable differentiation of highly pure cortical neurons from human induced pluripotent stem cells.

Human-induced-pluripotent-stem-cell (hiPSC)-derived neurons are valuable for investigating brain physiology and disease. Here, we present a protocol to differentiate hiPSCs into cortical neurons with high yield and purity. We describe neural induction via dual-SMAD inhibition, followed by spot-based differentiation to provide high quantities of neural precursors. We detail their enrichment, expansion, and purification to avoid unwanted cell fates and provide optimal conditions for neural rosette proliferation. These differentiated neurons are suitable for drug testing and co-culture studies. For complete details on the use and execution of this protocol, please refer to Paquet et al.1 and Weisheit et al..2.

TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1, and Batten disease.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Detection of Deleterious On-Target Effects after HDR-Mediated CRISPR Editing.

CRISPR genome editing is a promising tool for translational research but can cause undesired editing outcomes, both on target at the edited locus and off target at other genomic loci. Here, we investigate the occurrence of deleterious on-target effects (OnTEs) in human stem cells after insertion of disease-related mutations by homology-directed repair (HDR) and gene editing using non-homologous end joining (NHEJ). We identify large, mono-allelic genomic deletions and loss-of-heterozygosity escaping standard quality controls in up to 40% of edited clones. To reliably detect such events, we describe simple, low-cost, and broadly applicable quantitative genotyping PCR (qgPCR) and single-nucleotide polymorphism (SNP) genotyping-based tools and suggest their usage as additional quality controls after editing. This will help to ensure the integrity of edited loci and increase the reliability of CRISPR editing.