The misfolding and accumulation of tau protein into intracellular aggregates known as neurofibrillary tangles is a pathological hallmark of neurodegenerative diseases such as Alzheimer’s disease. However, while tau propagation is a known marker for disease progression, exactly how tau propagates from one cell to another and what mechanisms govern this spread are still unclear. Here, we report that cellular internalization of tau is regulated by quaternary structure and have developed a cellular assay to screen for genetic modulators of tau uptake. Using CRISPRi technology we have tested 3200 genes for their ability to regulate tau entry and identified enzymes in the heparan sulfate proteoglycan biosynthetic pathway as key regulators. We show that 6-O-sulfation is critical for tau-heparan sulfate interactions and that this modification regulates uptake in human central nervous system cell lines, iPS-derived neurons, and mouse brain slice culture. Together, these results suggest novel strategies to halt tau transmission.
Supporting Information: Figure S1 Figure. S1 -Endogenous tagging of endolysosomal compartments (A) Representative fluorescence microscopy images of CRISPRi-HEK293T cells with RAB7A-mNG 11 endogenously labeled with the split-mNeonGreen system. Cells were treated with AF555-tau fibrils for 22 hours. (B) Representative fluorescence microscopy images of HEK293T cells with mNG 11 -RAB7A (top) or LAMP1-mNG 11 (bottom) endogenously labeled with the split-mNeonGreen system.
Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.DOI:
http://dx.doi.org/10.7554/eLife.15802.001
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