JPND Genomic Instability in Alzheimer's Disease and Related Disorders: a Single-Cell Approach

Lead Research Organisation: The Wellcome Trust Sanger Institute
Department Name: Wellcome Trust Genome Campus

Abstract

Chromosomes are carriers of our genetic information, the human DNA. Human cells contain 23 pairs of chromosomes, collectively called the human genome, and express a specific set of genes from that genome via RNA molecules, collectively called the transcriptome. The human genome is believed to be stable throughout the development of a human individual. However, recent analyses of human single cells challenge this dogma. While novel genetic alterations acquired following conception can lie at the root of various genetic disorders, including cancer, amazingly little is known about the true rate at which somatic DNA-mutation occurs, the different natures of the acquired mutations, how it varies from cell type to cell type, its precise role in the development of different diseases, and how genome stability may be influenced by genetic background, aging, or other factors.

The general objective of this proposal is to study the role of neuronal genomic instability in the pathogenesis of Alzheimer's disease (AD) and related primary Tauopathies (a class of neurodegenerative diseases associated with the pathological aggregation of Tau protein in the brain) in order to provide significant insights and better diagnostic, preventive and therapeutic strategies. The proposal builds on preliminary findings highlighting a novel and direct role for Tau in genome stability and suggesting that somatic genomic variation acts as a causal player in neuropathology. The proposal is subdivided into four specific research objectives: (1) Determine the nature and extent of genomic and transcriptomic instability in human AD and primary Tauopathy brains; (2) Identify mosaic causal mutations in human AD and primary Tauopathy brains; (3) Study the role of Tau protein/pathology in DNA/RNA protection and damage; (4) Study the role of Tau protein/pathology in mitotic chromosomal instability. A multidisciplinary consortium that will use single-cell genome and transcriptome sequencing in human brain tissue as well as experimental Tauopathy-relevant models including mice and Drosophila will achieve these objectives. The project is particularly ambitious at the technological level as it plans to implement highly powerful and novel next-generation sequencing methodologies to single cells which is needed to address the groundbreaking objectives stated above. This proposal addresses the call "Genetic, epigenetic and environmental risk and protective factors for neurodegenerative diseases" and tackles the highly prevalent age-related brain diseases AD and related disorders characterized by Tau pathology. It aims at understanding how altered stability of the neuronal genome in the developing and adult brain determines the risk to develop these chronic disorders in late-adulthood. The expected impact is to genetically explain the pathogenesis of a consistent part of sporadic AD cases, for which no pathogenic cause is known yet and to decipher underlying molecular mechanisms that will lead to new early therapeutic tools.

Technical Summary

To study the nature and extent of genomic instability in human AD and primary Tauopathy brains, we will sequence the genomes of ~1500 single neurons obtained from frozen frontal cortex brain tissue of 10 normal, 10 AD, and 10 Tauopathy brains. Computational methods will reveal DNA-copy number landscapes and structural rearrangements from the single-cell sequences.

To study the nature and extent of transcriptomic instability in human AD and primary Tauopathy brains, we will apply single-cell resolution spatial transcriptomics to tissue sections of those same brains. Spatial transcriptomics enables molecular imaging of the full mRNA transcriptome in a tissue section by sequencing.

To identify somatic mutations in cells that may cause neuropathology, we will employ whole genome (and targeted) sequencing approaches on single neurons isolated from different areas of a subject's brain. Specifically, we will analyse subjects with no family history of AD or Tauopathies, and no causal germline mutation.

To determine the role of Tau/Tau pathology in (i) transcription regulation, we will apply spatial transcriptomics to brains of WT, KOTau and THY-Tau22 mice; and (ii) mitochondrial and genomic DNA integrity and repair, as well as (iii) chromatin organization, we will apply a diversity of techniques as e.g. immunostaining on brain.

To determine the molecular mechanism by which Tau induces genomic instability, we will (i) characterize the human Tau-induced mitotic aberrations in Drosophila larval neuroblasts and developing photoreceptor neurons, (ii) identify genetic suppressors of hTau-induced spindle defects and genomic instability in Drosophila, (iii) characterize the human Tau-induced mitotic aberrations in a Tau inducible neuroblastoma cell line, (iv) characterize human Tau-induced mitotic aberrations in embryonic brains in the humanized genomic Tau mouse model, and (v) subject brain tissue from Drosophila and mouse models to single cell genome sequencing.

Planned Impact

Note: the following text has been cut and pasted from the main application as requested.
1. Short-term: novel mechanistic insights: The INSTALZ project is expected to have a clear impact on our understanding of the pathogenesis of AD and related Tauopathies. The INSTALZ project will help to determine the cellular state that causes a particular but not the adjacent neuron to die in AD and related diseases. This is important and could explain why particular neurons and/or specific brain regions are more vulnerable than others, a longstanding and unexplained phenomenon in neurodegenerative brain diseases. In addition, since (1) somatic mutations may not be passed to children, depending on the developmental timing of the appearance of the genetic lesion, and (2) a mutation private to the brain would not be found in peripheral tissues, these somatic mutations would not be possible to identify during a "classical" genetic screen. The expected impact is to explain the pathogenesis of a consistent part of sporadic AD cases, for which no pathogenic cause (genetic or otherwise) is known yet, another longstanding and unexplained phenomenon in neurodegenerative brain diseases.
2. Medium-term: diagnostic or biomarker tests: Single-cell technologies already have a considerable impact on the development of novel diagnostic tools in medicine. For example, for patients with cancer, single cell technologies are playing an increasing role in the detection of minimal residual disease or in the analysis of circulating tumor cells in the peripheral blood. In the context of brain disease it is unlikely that invasive brain biopsy-based single-cell tests would become a routine diagnostic test. However, we anticipate that mechanisms leading to brain mosaicism are also active in peripheral proliferating tissues such as blood or skin and can likely be detected in these accessible tissues, e.g. buccal swabs. Given the enormous size of the problem of AD and neurodegenerative brain disease in our aging society, the market for the development of such biomarker tests in periperal tissues is huge.
3. Long-term: therapeutic applications: Our research will provide fundamental novel insights into the molecular mechanisms by which neurons degenerate in AD and Tauopathies. In the long-term therapeutic stategies aimed at the protection of genomically vulnerable neurons can be imagined.

Publications

10 25 50
 
Title Apply G&T-seq method on AD single nuclei 
Description We have now implemented a robust multi-omics single cell approach, abbreviated snG&T-seq, which allows to study the genome and transcriptome of the same single nuclei. This enables high accurate sequencing of single cell genomes as well as sequencing the polyadenylated transcripts contained within the same single nucleus. We have optimised this method on healthy neurons and currently we are studying genome instability in neurons from Tauopathy brain. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact This method will help to study heterogeneity in human brain in relation to disease at a single cell resolution. Due to the morphology of neurons in the cortex, other methods such as standard G&T-seq on intact cells was not suitable to apply on frozen post-mortem brain tissue. We will be applying this method on single neuronal nuclei derived from postmortem human brain samples and potentially animal models, to study Tauopathy and normal control brains obtained from brain banks or collaborators. 
 
Title Computational G&T-seq methodology to identify somatic heterogeneity and mutational landscape of single cells in brain 
Description We are refining computational methods to accurately analyse G&T-sequences; specifically genomic and transcriptomic data simultaneously derived from a single nucleus. This method will be applied on single-nuclei G&T-seq data from Tauopathy samples and controls. Layers of information from gDNA and cDNA will be collated and analysed jointly to reduce single cell-related technical noise and identify true variants. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2019 
Provided To Others? Yes  
Impact Ultimately, this method aims to produce an accurate landscape of genomic instability in Tauopathy disorders at single cell resolution. The result will lead to the identification of candidate causal variations and processes in neurodegenerative disorders. 
 
Title Dataset of single-nucleus genome and transcriptome sequences derived from human control neurons 
Description Dataset of single-nucleus genome and transcriptome sequences derived from control neurons of 5 human brains of different age categories. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact This dataset will be part of a publication on genetic variation in human neurons. 
 
Description CRJA Galas Lab 
Organisation University of Lille
Country France 
Sector Academic/University 
PI Contribution We obtained DNA libraries for sequencing from collaborators. Specifically, we are performing G&T-sequencing and data-analysis of cDNA and gDNA libraries produced from RNA and DNA isolated from the hippocampi of 1 KOtau & 1 WT littermate mouse as well as 1 THY-Tau22 mouse & 1 WT littermate mouse.
Collaborator Contribution The lab of Marie-Christine Galas provided single-cell genetic material isolated from 3 KOTau & 3 littermate WT mice as well as 3 THY-Tau22 mice & 3 littermate WT mice.
Impact We obtained copy number profiles from this analysis, no major genomic instability was detected in this data. This collaboration is not multi-disciplinary.
Start Year 2016
 
Description Cambridge Brain Bank 
Organisation Cambridge University Hospitals NHS Foundation Trust
Department Cambridge Brain Bank
Country United Kingdom 
Sector Public 
PI Contribution RNA integrity score of tauopathy donor brains collected from the Cambridge Brain Bank appear too low for single-nucleus genome-plus-transcriptome sequencing (20 tauopathy samples tested plus 20 normal control samples).
Collaborator Contribution Providing of samples.
Impact No outcome. This was planned as multi-disciplinary research (clinicians, molecular biology, bioinformatics).
Start Year 2018
 
Description IPL Dermaut Lab 
Organisation Pasteur Institute, Lille
Country France 
Sector Charity/Non Profit 
PI Contribution We processed 5 control brains of the 44 brains obtained from the Lille neurobank further with single nucleus G&T-seq, which were originally screened by IPL .
Collaborator Contribution The IPL-Dermaut lab performed bulk gDNA isolation from unaffected brain tissue of the 44 brains obtained from the Lille Neurobank for constitutional mutation screening and H1/H2 haplotyping. As well, the IPL-Dermaut lab provides Drosophila-Tau lines.
Impact We received the results of the constitutional mutation screening and H1/H2 haplotyping for the brains which were further processed with snG&T-seq.
Start Year 2016
 
Description Identifying low frequency somatic variants in AD brain samples 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution This project is a joint collaboration with a member of the team: Dr. Raheleh Rahbari is collaborating with Dr. Carlo Sala Frigerio from Prof. Bart De Strooper's group to develop and implement statistical models for the identification of low frequency somatic variants in AD brain samples.
Collaborator Contribution This collaboration is a joint effort to discriminate true positive variants with low allele frequency from sequencing errors. Our partners have performed targeted deep sequencing of several AD brain samples. Currently we are developing methods that produce a comprehensive error model per site and identifies candidates with high probability of being true positive variants.
Impact This work is in progress. This collaboration is not multi-disciplinary.
Start Year 2017
 
Description KU Leuven 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution Sequencing and analysis of genetic material from single cells obtained from the collaborator.
Collaborator Contribution Separation and amplification of DNA and RNA from lysed single nuclei, and preparation of cDNA and gDNA libraries for sequencing.
Impact We have obtained genetic material of single nuclei from this collaborator. We have performed single nucleus genome-plus-transcriptome sequencing and analysis of this genetic material. The ongoing analysis showed genome instability in a proportion of healthy neurons from human prefrontal cortex. This collaboration is not multi-disciplinary.
Start Year 2016
 
Description Lille Neurobank & KU Leuven 
Organisation Lille University Hospital
Country France 
Sector Hospitals 
PI Contribution We performed sequencing and analysis of genetic material from single nuclei obtained from the collaborator.
Collaborator Contribution The Lille Neurobank provides frontal cortex tissue to KU Leuven for single cell processing and unaffected tissue from occipital cortex or cerebellum to IPL for constitutional mutation screening from 44 brains (10 PSP, 3 CBD, 6 FTDP-17, 19 AD and 6 normal control). RNA Integrity Scores of Tauopathy donor brains collected from Lille Neurobank (36 brains tested) appear too low for single-nucleus genome-plus-transcriptome sequencing (snG&T-seq).
Impact The ongoing analysis showed genome instability in a proportion of healthy neurons from human prefrontal cortex. We identified a contamination in the RNA-sequences of part of the cells, which is currently being resolved. This collaboration is multi-disciplinary. (Molecular biology, bio-informatics & clinicians)
Start Year 2016
 
Description UAntwerp-Institute Born-Bunge Biobank and KU Leuven 
Organisation University of Antwerp
Country Belgium 
Sector Academic/University 
PI Contribution Ethics documents have been completed. The Material transfer agreement is being finalized for transfer of sorted neuronal nuclei (DNA/RNA) of tauopathies collected from the Antwerp Brain Bank from KU Leuven to Sanger
Collaborator Contribution KU Leuven has identified high quality tauopathy brain samples with high RNA integrity scores from the UAntwerp-Institute Born-Bunge Biobank, and has previously obtained the required ethics and agreement documentation.
Impact No outcome yet. This collaboration is multi-disciplinary (clinicians, molecular biology, bioinformatics).
Start Year 2019