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

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.

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.

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.