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A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death

Abstract

We have identified a rare coding mutation, T835M (rs137875858), in the UNC5C netrin receptor gene that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer's disease and that was associated with disease across four large case-control cohorts (odds ratio = 2.15, Pmeta = 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and in vitro studies demonstrate that this mutation leads to increased cell death in human HEK293T cells and in rodent neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to cell death from multiple neurotoxic stimuli, including β-amyloid (Aβ), glutamate and staurosporine. On the basis of these data and the enriched hippocampal expression of UNC5C in the adult nervous system, we propose that one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer's disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer's disease brain.

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Figure 1: Identification of T835M UNC5C as a candidate variant for LOAD.
Figure 2: Association of T835M UNC5C (rs137875858) in AD case and control cohorts.
Figure 3: T835M UNC5C increases cell death beyond that induced by WT UNC5C.
Figure 4: UNC5C is expressed in the adult hippocampus and the T835M UNC5C variant sensitizes hippocampal neurons to neurotoxic stimuli.

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GenBank/EMBL/DDBJ

NCBI Reference Sequence

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Acknowledgements

This work was partially supported by grants from the US National Institutes of Health (R01-AG044546, P50-AG05681), and the Alzheimer's Association (NIRG-11-200110). This research was conducted while C.C. was a recipient of a New Investigator Award in Alzheimer's disease from the American Federation for Aging Research. C.C. is a recipient of a BrightFocus Foundation Alzheimer's Disease Research grant (A2013359S). Samples from the National Cell Repository for Alzheimer's Disease (NCRAD), which receives government support under a cooperative agreement grant (U24 AG21886) awarded by the National Institute on Aging (NIA), were used in this study. NIA-LOAD samples were collected under a cooperative agreement grant (U24 AG026395) awarded by the NIA. The ADGC is funded by the NIA (UO1AG032984). We thank K. Steffansøn and DeCode Genetics for genotype counts of UNC5C variants in the Icelandic population, the Alzheimer's Disease Centers, who collected samples used in this study, and patients and their families, whose help and participation made this work possible. We thank J. Norton of the Genetics Core, Washington University. We also thank C. Nelson (Genentech) for the HEK293 wild-type human APP695 stable cell line, K. Hoyte, Y. Lu, M. Sagolla, L. Gilmor, J. Borneo, E. Ladi, J. Grogan, J. Larson and J. Kaminker for technical assistance, and S. Ackerman, The Jackson Laboratory/Howard Hughes Medical Institute, for the generous gift of Unc5c−/− mice.

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M.K.W.-S. and J. Hunkapiller designed, conducted and analyzed biological experiments and wrote the manuscript, T.R.B. performed linkage, case-control association, survival, growth-curve and sequencing data analysis, K.S. developed a method for isolating and sorting cell-type-specific adult brain for quantitative PCR, J.A.M. generated vectors and contributed to in vitro experiments, J.K.A. supervised the project, S.M.S. performed Unc5c in situ hybridization (ISH), M.B.Y. performed UNC5C ISH, O.F. contributed to ISH experiments and generated images, N.R. performed sample genotyping and handling and contributed to the manuscript, W.O. coordinated DNA sample collection, sequencing and data management, D.V.H. supervised development of the method for isolating and sorting cell-type-specific adult brain for quantitative PCR, M.T.-L. contributed to the manuscript, the ADGC provided AD case and control cohorts, R.M. provided sample material and contributed to the manuscript, M. P.-V., J. Haines, L.A.F., G.D.S. and A.G. provided sample material and contributed to the manuscript, T.W.B., C.C. and R.J.W. supervised the project and contributed to the manuscript, and R.R.G. supervised the project and wrote the manuscript.

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Correspondence to Carlos Cruchaga or Ryan J Watts or Robert R Graham.

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M.K.W.-S., J.H., T.R.B., K.S., J.A.M., J.K.A., S.M.S., M.B.Y., O.F., W.O., N.R., D.V.H., T.W.B., R.J.W. and R.R.G. are full-time employees of Genentech.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14, Supplementary Tables 1–4 and Supplementary Results (PDF 131708 kb)

Live-cell imaging movies of cell death and caspase-3 or 7 activation in real time of β-gal-transfected HEK293 cells.

Representative live-cell imaging movie of β-gal-displaying transfected cells (red) and those cells with caspase-3 or 7 activity (green). (MOV 274 kb)

Live-cell imaging movies of cell death and caspase-3 or caspase-7 activation in real time of WT UNC5C-transfected HEK293 cells.

Representative live-cell imaging movie of WT UNC5C-displaying transfected cells (red) and those cells with caspase-3 or caspase-7 activity (green). (MOV 181 kb)

Live-cell imaging movies of cell death and caspase-3 or caspase-7 activation in real time of T835M UNC5C-transfected HEK293 cells.

Representative live-cell imaging movie of T835M UNC5C displaying transfected cells (red) and those cells with caspase-3 or 7 activity (green). (MOV 152 kb)

Live-cell imaging movies of β-gal-expressing hippocampal neurons.

Representative live-cell imaging movie of β-gal-expressing neurons exhibiting GFP+ transduced cells (green), as expression is initiated, maintained or lost as neurons undergo basal cell death. (MOV 2431 kb)

Live-cell imaging movies of WT UNC5C-expressing hippocampal neurons.

Representative live-cell imaging movie of WT UNC5C-expressing neurons exhibiting GFP+ transduced cells (green), as expression is initiated, maintained or lost as neurons undergo basal cell death. (MOV 2364 kb)

Live-cell imaging movies of T835M UNC5C-expressing hippocampal neurons undergoing basal cell death.

Representative live-cell imaging movie of T835M UNC5C-expressing neurons exhibiting GFP+ transduced cells (green), as expression is initiated, maintained or lost as neurons undergo basal cell death. (MOV 2328 kb)

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Wetzel-Smith, M., Hunkapiller, J., Bhangale, T. et al. A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death. Nat Med 20, 1452–1457 (2014). https://doi.org/10.1038/nm.3736

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