Periodic Reporting for period 4 - REBUILDCNS (Redirecting glial progenitor fate to rebuild the injured Brain )
Reporting period: 2022-12-01 to 2023-05-31
To increase the quality of life of our aging population is a long-term goal for
our society. Cancer, stroke and other cerebrovascular accidents are among
the leading causes of death, cognitive impairment and degradation of quality
of life. We use clinically relevant animal models to understand the molecular
principles that govern the capacity of the brain to self-repair. Once we
understand these principles, candidate molecules will emerge as therapeutic
targets to regenerate the diseased brain. A key aspect of our research is the
development and application of new technologies to study and manipulate
rare stem cells in their native environment. As an example, we have established
a method to study DNA-demethylation from small number or single cells as well
as a nuclease-based system for epigenome editing in vivo.
Similar to our already developed single cell analyses, these technologies can
be applied far beyond neuronal stem cells, and our leading expertise will be
very valuable to the Scientific community studying stem cells in adult tissues.
our society. Cancer, stroke and other cerebrovascular accidents are among
the leading causes of death, cognitive impairment and degradation of quality
of life. We use clinically relevant animal models to understand the molecular
principles that govern the capacity of the brain to self-repair. Once we
understand these principles, candidate molecules will emerge as therapeutic
targets to regenerate the diseased brain. A key aspect of our research is the
development and application of new technologies to study and manipulate
rare stem cells in their native environment. As an example, we have established
a method to study DNA-demethylation from small number or single cells as well
as a nuclease-based system for epigenome editing in vivo.
Similar to our already developed single cell analyses, these technologies can
be applied far beyond neuronal stem cells, and our leading expertise will be
very valuable to the Scientific community studying stem cells in adult tissues.
To understand the principles that govern the capacity of endogenous neural stem cells (NSCs) to repair the brain, we first established the required molecular tools. To be able to interrogate a high number of stem cells simultaneously at an affordable cost and time, we have implemented and miniaturized SMART-Seq3 for single RNA sequencing and single cell sequencing of the nucleosome, methylome and transcriptome (scNMT).
The SMART-Seq3 protocol has a key advantage over the conventional SMART-Seq2 technology, namely, that through the "unique modyfied identifiers" (UMI) it allows quantitative analysis of single cell transcriptome. The scNMT technology replaces the initiatlly proposed whole genome bisulfite sequencing (WGBS) of low input bulk-samples. It additionally allows parallel analysis of the nucleosome, methylome and transcriptome from the same cell. Therefore, this technology will provide unprecedent insights on the control of the methylome code over the transcriptome at the single cell level, which is one of the main aims of our project. We have already shown, that we are able to identify differentially methylated regions (DMRs) between neurons, stem cells and neuroblasts using the scNMT technology. These DMRs appear to be largely independent of chromatin accessibility. The high resolution of single cell technology has given us the ability to compare DNA methylation with the expression of nearby genes, allowing prediction of possible target genes of DMRs or other genomic regions such as enhancers.
On the other side, we established tools to selectively target adult neural stem cells within the subventricular zone. To this end, we use adeno-associated-virus, as they are the safest alternative for gene therapy in humans. To identify the best capsid variant to selectively transduced these cells we conducted a high-throughput screening of 177 capsid known and novel variants. These screening identified two main capsid variants targeting adult neural stem cells. This study is published in Molecular Therapy Methods and Clinical Development (Kremer et al., 2021).
These novel capsid variants will be used to establish lineage tracing. The initially proposed strategy of using avian sarcoma-leukosis virus (ASLV)-barcoded library for lineage tracing, had to be discarded, due to malformations of the subventricular zone of Nestin-TVA-tdTomato mice. We thus were establishing an adeno-associated virus (AAV)-based SCAR tracing method. This is based on the introduction of INDELs (barcodes) by CRISPR-Cas9, which can then be recovered using scRNA-seq, in the Rosa26:tdRFP locus in adult mice. Due to technical limitations of this mouse line we decided to replace it by the ROSA26:mGFP mouse line. This mouse line expresses membrane GFP constitutively in virtually all cells. Upon SaCas9 endonuclease activity, indels (barcodes) will be permanently introduced in the mGFP locus and will abolish GFP fluorescence in NSCs, allowing their identification and sorting for single cell RNA-seq. The identification of barcodes will allow the readout of clonal relationships among cells derived from the same NSC.The first step for this implementation consisted on selecting the optimal gRNA (or combination of gRNAs) to best target the CRISPR machinery to the mGFP locus. Main criteria in the optimization process were a high loss of fluorescence and a high variability of mutations generated.
In summary, our proposed methodology proved useful to perform clonal lineage tracing studies in the adult SVZ. Our most immediate aim will be to achieve a higher NSC-targeting at the injection time. In the longer run, we would like to study the clonal properties of the NSC niche both in homeostasis, ageing and upon injury.
The generated data will be used as ground truth for the development of an manipulation-free lineage tracing computational tool based on so-called single nucleotide polymorphisms (SNPs). SNPs are spontaneously occurring mutations that arise in a cell and are thus passed onto its progeny.
To study the molecular link between transcriptome and epigenome in glial progenitors during homeostasis we used single cell NMT method. These data shed light on the association of specific DNA methylation profiles with discrete astrocyte categories: the prevalent astrocyte and the specialized neural stem cells.
Interferons play an important role during homeostasis and injury/ageing in neural stem cells.In the old brain interferons contribute to a decline in neural stem cell function. However, the role of interferons in the young brain is not well understood. Our aim was to understand the presence and impact of interferon regulation in the yound and aged brain. One important finding was that interferons exhibited a selective influence on neural stem cells, distinct from their effects on neural progenitors. This was detectable in young and old brains. To unravel the molecular mechanisms uderlying the interferon response a comprehensive analysis was conducted, including cell cycle progression, transcriptome, translatome and phospho-proteome of neural stem cells exposed to interferon β. A key revelation was that interferon β triggered a transient activation of mTORC1 while simultaneously inducing a cell cycle arrest, leading neural stem cells into a quiescent G0 state. Importantly, this interferon-mediated uncoupling of mTORC1 and the cell cycle resulted in the repression of Sox2, a pivotal factor in stem cell activity. Furthermore, interferon β exhibited a biphasic effect on mTORC1 activity, coupled with an inhibition of the cell cycle, ultimately promoting the exit of neural stem cells from their activation state (Carvajal-Ibanez et al., 2023).
The SMART-Seq3 protocol has a key advantage over the conventional SMART-Seq2 technology, namely, that through the "unique modyfied identifiers" (UMI) it allows quantitative analysis of single cell transcriptome. The scNMT technology replaces the initiatlly proposed whole genome bisulfite sequencing (WGBS) of low input bulk-samples. It additionally allows parallel analysis of the nucleosome, methylome and transcriptome from the same cell. Therefore, this technology will provide unprecedent insights on the control of the methylome code over the transcriptome at the single cell level, which is one of the main aims of our project. We have already shown, that we are able to identify differentially methylated regions (DMRs) between neurons, stem cells and neuroblasts using the scNMT technology. These DMRs appear to be largely independent of chromatin accessibility. The high resolution of single cell technology has given us the ability to compare DNA methylation with the expression of nearby genes, allowing prediction of possible target genes of DMRs or other genomic regions such as enhancers.
On the other side, we established tools to selectively target adult neural stem cells within the subventricular zone. To this end, we use adeno-associated-virus, as they are the safest alternative for gene therapy in humans. To identify the best capsid variant to selectively transduced these cells we conducted a high-throughput screening of 177 capsid known and novel variants. These screening identified two main capsid variants targeting adult neural stem cells. This study is published in Molecular Therapy Methods and Clinical Development (Kremer et al., 2021).
These novel capsid variants will be used to establish lineage tracing. The initially proposed strategy of using avian sarcoma-leukosis virus (ASLV)-barcoded library for lineage tracing, had to be discarded, due to malformations of the subventricular zone of Nestin-TVA-tdTomato mice. We thus were establishing an adeno-associated virus (AAV)-based SCAR tracing method. This is based on the introduction of INDELs (barcodes) by CRISPR-Cas9, which can then be recovered using scRNA-seq, in the Rosa26:tdRFP locus in adult mice. Due to technical limitations of this mouse line we decided to replace it by the ROSA26:mGFP mouse line. This mouse line expresses membrane GFP constitutively in virtually all cells. Upon SaCas9 endonuclease activity, indels (barcodes) will be permanently introduced in the mGFP locus and will abolish GFP fluorescence in NSCs, allowing their identification and sorting for single cell RNA-seq. The identification of barcodes will allow the readout of clonal relationships among cells derived from the same NSC.The first step for this implementation consisted on selecting the optimal gRNA (or combination of gRNAs) to best target the CRISPR machinery to the mGFP locus. Main criteria in the optimization process were a high loss of fluorescence and a high variability of mutations generated.
In summary, our proposed methodology proved useful to perform clonal lineage tracing studies in the adult SVZ. Our most immediate aim will be to achieve a higher NSC-targeting at the injection time. In the longer run, we would like to study the clonal properties of the NSC niche both in homeostasis, ageing and upon injury.
The generated data will be used as ground truth for the development of an manipulation-free lineage tracing computational tool based on so-called single nucleotide polymorphisms (SNPs). SNPs are spontaneously occurring mutations that arise in a cell and are thus passed onto its progeny.
To study the molecular link between transcriptome and epigenome in glial progenitors during homeostasis we used single cell NMT method. These data shed light on the association of specific DNA methylation profiles with discrete astrocyte categories: the prevalent astrocyte and the specialized neural stem cells.
Interferons play an important role during homeostasis and injury/ageing in neural stem cells.In the old brain interferons contribute to a decline in neural stem cell function. However, the role of interferons in the young brain is not well understood. Our aim was to understand the presence and impact of interferon regulation in the yound and aged brain. One important finding was that interferons exhibited a selective influence on neural stem cells, distinct from their effects on neural progenitors. This was detectable in young and old brains. To unravel the molecular mechanisms uderlying the interferon response a comprehensive analysis was conducted, including cell cycle progression, transcriptome, translatome and phospho-proteome of neural stem cells exposed to interferon β. A key revelation was that interferon β triggered a transient activation of mTORC1 while simultaneously inducing a cell cycle arrest, leading neural stem cells into a quiescent G0 state. Importantly, this interferon-mediated uncoupling of mTORC1 and the cell cycle resulted in the repression of Sox2, a pivotal factor in stem cell activity. Furthermore, interferon β exhibited a biphasic effect on mTORC1 activity, coupled with an inhibition of the cell cycle, ultimately promoting the exit of neural stem cells from their activation state (Carvajal-Ibanez et al., 2023).
Implementation of miniaturized SS3 and scNMT protocol for 384 cells
In addition, through the SCAR tracing method, we will establish an elegant AAV-based method to track the stem cell lineages in vivo. In the long run, these methods will provide a tremendous gain in knowledge in the field of neural stem cell activation under homeostatic, injury and aging conditions, thus providing new approaches for potential therapies.
In addition, through the SCAR tracing method, we will establish an elegant AAV-based method to track the stem cell lineages in vivo. In the long run, these methods will provide a tremendous gain in knowledge in the field of neural stem cell activation under homeostatic, injury and aging conditions, thus providing new approaches for potential therapies.