Tjernberg, L. O. | Search bioRxiv

bioRxiv2021

Engineered human induced pluripotent cells enable genetic code expansion in brain organoids

Human induced pluripotent stem cell (hiPSC) technology has revolutionized human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to amber stop codons. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/449576v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4e1fa6org.highwire.dtl.DTLVardef@fff373org.highwire.dtl.DTLVardef@1c839e6org.highwire.dtl.DTLVardef@5150e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Distinctive alteration of presynaptic proteins in the outer molecular layer of the dentate gyrus in Alzheimer's disease

Synaptic degeneration has been reported as one of the best pathological correlate of cognitive deficit in Alzheimers Disease (AD). However, the location of these synaptic alterations within hippocampal sub-regions, the vulnerability of the presynaptic versus postsynaptic compartments, and the biological mechanisms for these impairments remain unknown. Here, we performed immunofluorescence labeling of different synaptic proteins in fixed and paraffin embedded human hippocampal sections and report reduced levels of several presynaptic proteins of the neurotransmitter release machinery (complexin-1, syntaxin-1A, synaptotagmin-1 and synaptogyrin-1) in AD cases. The deficit was restricted to the outer molecular layer (OML) of the dentate gyrus whereas other hippocampal sub-fields were preserved. Interestingly, standard markers of postsynaptic densities (SHANK2) and dendrites (MAP2) were unaltered, as well as the relative number of granule cells in the dentate gyrus, indicating that the deficit is preferentially presynaptic. Notably, staining for the axonal components, myelin basic protein, SMI-312 and Tau, was unaffected, suggesting that the local presynaptic impairment does not result from axonal loss or alterations of structural proteins of axons. There was no correlation between the reduction in presynaptic proteins in OML and the extent of the amyloid load or of the dystrophic neurites expressing phosphorylated forms of Tau. Altogether, this study highlights the distinctive vulnerability of the OML of dentate gyrus and supports the notion of presynaptic failure in AD.

neuroscience↗