Alzheimer?s disease (AD) is the leading cause of dementia in the elderly and sixth leading cause of death in the US, and much risk remains unexplained. Brain somatic mosaicism (BSM) refers to the accumulation of mutations within CNS cells, occurring during development and aging, in two classes: clonal (i.e. one mutation present in two or more postmitotic cells) or non-clonal (a mutation uniquely present in only one postmitotic cell). The extent, impact and mechanisms of BSM on neurodegeneration remain mostly unexplored. Prior work from the NIH- funded Brain Somatic Mosaicism Network (BSMN) made critical breakthroughs in the ability to reliably assess BSM in bulk tissue and single nuclei, but also raised new questions regarding the extent to which BSM contributes to neurodegenerative disease. Recent studies from the labs of the PIs and others identified evidence of BSM contributing to neurological disease, and that neurons contain thousands of somatic mutations. Moreover, neurons in certain childhood progeroid neurodegenerative conditions accumulate mutations at twice the control rate. We hypothesize AD risk and disease progression is in part related to an excessive burden of neuronal somatic mutations. Here we propose an exploratory high-impact study to assess whether individual neurons from APOE e3/e3 AD and controls can be quantitatively assessed for burden of somatic mutations, and whether AD neurons display a greater burden of mutations. We have assembled a stellar scientific team representing expertise in neurogenetics, single cell genomics, and AD. We will utilize AD and control cases from the exceptional Shiley-Marcos Alzheimer?s Disease Research Center (ADRC) for study. We have acquired the following preliminary data: 1] Developed and extensive optimized methods for Fluorescent Activated Nuclear Sorting (FANS) NeuN+ PFC single neuronal nuclei from brain. 2] Developed single nuclear whole genome amplification (snWGA) methods to perform single nuclear whole genome sequencing (snWGS). 3] Optimized methods for high-throughput confirmation of BSM mutations. 4] Developed computational methods to assess quantitative differences in BSM rates. We propose aims to assess our hypotheses. 1] Perform snWGS on 30 NeuN+ FANS sorted nuclei on each of six AD cases and six age- and sex-matched controls. 2] Assess in a blinded fashion the extent of BSM mutations in the aged brain. 3] Compare accumulated BSM mutations in AD vs. control. 4] Profile mosaic mutations based upon their mutational signatures and clonality. These data will allow us to assess for the first time whether neurons in AD show evidence of an excess accumulation of somatic mutations. If our hypothesis is confirmed, in future proposals we will assess: 1] Whether BSM mutations correlate with AD genetic factors such as APOE e4. 2] Whether BSM mutations are also accumulated in other brain cell types in AD. 3] Whether other neuropathological forms of AD (i.e. with a-synuclein or TDP43 inclusions) also show BSM burden.
This proposal seeks to test the hypothesis, using cutting edge snWGS technology, that there is a greater degree of accumulation of somatic mutations in neurons of AD compared with age, sex and APOE genotype- matched controls.
