The identification of an expanding number of genetic susceptibility loci for Alzheimer's disease (AD) provides an opportunity for new mechanistic and therapeutic insights. The overall goal of this proposal is to bring together new genome engineering and stem cell technology to further our understanding of AD genetics and pathogenesis. The CRISPR-Cas9 system is a novel and facile platform for genome engineering that was described by us in the past year. We propose using the CRISPR-Cas system to generate a library of isogenic human IPSC lines with AD risk variants in each of the 10 susceptibility genes identified in multi-cohort GWAS studies. We have established 14 IPSC lines from dermal fibroblasts of late-onset AD (LOAD) and age-matched controls that have been differentiated to neurons and astrocytes. This led to the observation that IPSC-derived neural progenitors from LOAD patients differentiate to neurons prematurely, and differ in their transcriptional profiles when compared with controls. A similar phenotype was observed in AD vs control IPSC-derived neural progenitor lines from another laboratory. Informatic analysis implicated downregulation of a gene network controlled by the neural transcription factor REST/NRSF in AD neural progenitors. A similar change in this gene network was observed in vivo, in the brains of patients with mild cognitive impairment (MCI) and AD. Our preliminary studies suggest that REST also regulates genes involved in cell death pathways, AD pathology and inflammatory/immune responses. The generation of isogenic IPSC lines that differ only in the presence or absence of genetic variants associated with AD will enable us to explore the role of REST in a highly controlled manner, as well as investigate other pathogenic mechanisms involving A? metabolism, tau phosphorylation and neuronal stress responses. Furthermore, isogenic IPSCs will be differentiated into astrocytes and microglia to explore inflammatory phenotypes associated with risk alleles in the immune-related genes TREM- 2, CD33 and CR1. To elucidate the affected pathways, new high-sensitivity transcriptome sequencing and proteomic technology will be applied to IPSC-derived neurons and microglia with AD variants. Transcriptome data from isogenic cells will be integrated with RNA-seq analysis of the brain in subjects from the Religious Orders Study with the same genotypes. This approach will identify core regulatory mediators and pathways, with the goal of constructing a network model of AD risk variants. These studies will bring together two principal investigators and many collaborators with diverse but complementary areas of expertise in a multidisciplinary approach to understand genetic modifiers of AD.

Public Health Relevance

We have advanced a new platform for genome engineering of human stem cells. Using this technology, we will generate human neurons with identical genetic backgrounds that differ only in the presence of genetic changes that confer risk for Alzheimer's disease. This invaluable resource may lead to the discovery of novel disease pathways and therapeutic targets for Alzheimer's disease.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Multi-Year Funded Research Project Grant (RF1)
Project #
1RF1AG048056-01
Application #
8756257
Study Section
Special Emphasis Panel (ZAG1-ZIJ-5 (M1))
Program Officer
Wise, Bradley C
Project Start
2014-08-15
Project End
2019-07-31
Budget Start
2014-08-15
Budget End
2019-07-31
Support Year
1
Fiscal Year
2014
Total Cost
$2,095,914
Indirect Cost
$814,114
Name
Harvard Medical School
Department
Genetics
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Lin, Yuan-Ta; Seo, Jinsoo; Gao, Fan et al. (2018) APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron 98:1141-1154.e7
Lin, Yuan-Ta; Seo, Jinsoo; Gao, Fan et al. (2018) APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron 98:1294
Chari, Raj; Yeo, Nan Cher; Chavez, Alejandro et al. (2017) sgRNA Scorer 2.0: A Species-Independent Model To Predict CRISPR/Cas9 Activity. ACS Synth Biol 6:902-904
Lajoie, M J; Söll, D; Church, G M (2016) Overcoming Challenges in Engineering the Genetic Code. J Mol Biol 428:1004-21
Kang, Chanhee; Xu, Qikai; Martin, Timothy D et al. (2015) The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4. Science 349:aaa5612
Byrne, Susan M; Ortiz, Luis; Mali, Prashant et al. (2015) Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res 43:e21
Busskamp, Volker; Lewis, Nathan E; Guye, Patrick et al. (2014) Rapid neurogenesis through transcriptional activation in human stem cells. Mol Syst Biol 10:760