Periodic Reporting for period 3 - CELLPHASE_AD (Genetics to understand cellular components of Alzheimer Disease pathogenesis)
Reporting period: 2022-11-01 to 2024-04-30
Alzheimer disease (AD) is a major health problem worldwide. To develop new therapies we need to accelerate the translation of genetic risk genes into mechanistic insights. Many of the novel genetic risk factors for AD are expressed in microglia and astroglia, which remains an understudied population in this classically neuron-centric field. Human stem cells (iPSCs) provide great possibilities but are largely investigated in vitro, which does not capture the complexity of a human brain. On the other hand, promising results from our current rodent models also translate poorly into the clinic, highlighting the need for optimized models if we want to further understand this complex disease and advance the development of novel drugs.
We propose here the use of mouse-human xenotransplantation models to test the effects of AD-associated genetic risk factors on the phenotypes of transplanted microglia and astroglia derived from patients and from genomic engineered, isogenic stem cells. The cells will be followed during disease progression in the brain of wild type and of mice developing Aβ- and Tau-pathology, the pathological features of AD. Using single cell transcriptomics, a dynamic view of the cell states is generated over time.
In a first arm of the project, we investigate how the genetic makeup of patient derived stem cells with high and low polygenic risk scores influences pathological cell states. In the second arm of the project, we generate inducible CRISPR/CAS9 iPS isogenic cell lines to manipulate rapidly and specifically the expression of four selected AD-associated genes linked to a putative cholesterol pathway but also affecting inflammation. These cell lines will be used also in the second phase of the project when validating hypotheses generated from the extensive bioinformatics analysis of the 600.000 single human cell profiles generated.
We propose here the use of mouse-human xenotransplantation models to test the effects of AD-associated genetic risk factors on the phenotypes of transplanted microglia and astroglia derived from patients and from genomic engineered, isogenic stem cells. The cells will be followed during disease progression in the brain of wild type and of mice developing Aβ- and Tau-pathology, the pathological features of AD. Using single cell transcriptomics, a dynamic view of the cell states is generated over time.
In a first arm of the project, we investigate how the genetic makeup of patient derived stem cells with high and low polygenic risk scores influences pathological cell states. In the second arm of the project, we generate inducible CRISPR/CAS9 iPS isogenic cell lines to manipulate rapidly and specifically the expression of four selected AD-associated genes linked to a putative cholesterol pathway but also affecting inflammation. These cell lines will be used also in the second phase of the project when validating hypotheses generated from the extensive bioinformatics analysis of the 600.000 single human cell profiles generated.
The central hypothesis of this project is that a high genetic risk profile for AD promotes cell states in microglia and astroglia that contribute to disease, while a low genetic risk profile will stabilize cells in protective states. We are investigating genetic risk both from a single gene approach as well as from a polygenic risk approach (looking at the entire genetic profile). To this extent we have successfully generated stem cells in which specific risk genes for AD have been deleted, we have obtained stem cells isogenic for APOE, the largest AD risk gene, and we have obtained various stem cells with either a high or low polygenic risk score.
We have optimized specific protocols for generating stem cell-derived human astroglia and microglia and xenotransplanted these cells into the brain of mice developing amyloid-beta pathology and assessed whether specific genetic risk would affect the response of these cell types to Abeta pathology. Using single cell RNA sequencing we have explored the change in cell states that these cells adopt when being confronted with Abeta pathology. We have generated a unique catalogue of the response of 129.000 human microglia, identifying specific cell states that are only observed in human microglia and not in mouse microglia and the specific trajectories that these human microglia adopt when becoming activated. We also show that these responses can be modulated either by the genotype of the microglia as well as by the extent of Abeta pathology in the brain. A manuscript encompassing this data is currently being drafted.
Furthermore, we have also demonstrated that human astrocytes integrate into the mouse brain, adopt human-specific morphologies and electrophysiological properties, but that their APOE genotype does not affect their morphological response to Abeta pathology. This data was published in 2021 (Preman et al. 2021 Molecular Neurodegeneration 68).
Additional work is currently ongoing to investigate how polygenic risk can affect the microglial response to Abeta pathology and whether high polygenic risk indeed locks microglia in detrimental cellular states.
We have optimized specific protocols for generating stem cell-derived human astroglia and microglia and xenotransplanted these cells into the brain of mice developing amyloid-beta pathology and assessed whether specific genetic risk would affect the response of these cell types to Abeta pathology. Using single cell RNA sequencing we have explored the change in cell states that these cells adopt when being confronted with Abeta pathology. We have generated a unique catalogue of the response of 129.000 human microglia, identifying specific cell states that are only observed in human microglia and not in mouse microglia and the specific trajectories that these human microglia adopt when becoming activated. We also show that these responses can be modulated either by the genotype of the microglia as well as by the extent of Abeta pathology in the brain. A manuscript encompassing this data is currently being drafted.
Furthermore, we have also demonstrated that human astrocytes integrate into the mouse brain, adopt human-specific morphologies and electrophysiological properties, but that their APOE genotype does not affect their morphological response to Abeta pathology. This data was published in 2021 (Preman et al. 2021 Molecular Neurodegeneration 68).
Additional work is currently ongoing to investigate how polygenic risk can affect the microglial response to Abeta pathology and whether high polygenic risk indeed locks microglia in detrimental cellular states.
We expect to identify and validate >5 novel drug targets in the astroglia-microglia axis of AD pathogenesis.
Our work will provide humanized models for AD, an answer on how genetic makeup affects microglia and astroglia in an AD relevant context, and establish a highly versatile platform to explore human genetics in human cells in vivo.
Our work will provide humanized models for AD, an answer on how genetic makeup affects microglia and astroglia in an AD relevant context, and establish a highly versatile platform to explore human genetics in human cells in vivo.