Pharmacogenomic identification of small molecules for lineage specific manipulation of subventricular zone germinal activity

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Key issues and objectives of the thesis

One of the major challenge of regenerative medicine is to trigger tissue repair by recruiting endogenous stem cells. However, the efficiency of this approach in vivo remains largely limited. First, the endogenous stem cells may have lost their competency to produce the damaged cells. Second, if/when they differentiate correctly, they need to migrate toward the site of injury. Eventually, the microenvironment at the lesion site has to be permissive enough to allow a correct the integration of the newborn cells. Chronic hypoxia is a model of mild cortical injury followed by a
spontaneous cellular regeneration. Although a few studies focused on cortical oligodendrocyte and GABAergic neurons repair (Fagel et al., 2006; Jablonska et al., 2012), none focused on the source of glutamatergic neurons born after the hypoxic period. Postnatal subventricular zone stem cells represent a potential cellular source for cortical repair following injury. The most translational way to promote their participation to tissue repair is to use a pharmacological approach, i.e. small molecules, to increase their activity and trigger cellular recruitment and integration at the site of injury. Timely controlled modulation of signaling pathways is necessary to trigger neuronal specification and oligodendrocyte maturation. The selection of relevant small molecules will therefore depend on finding a tight balance between its ability to promote dorsal SVZ stem cell proliferation, trigger specific fate but also promote or at least not prevent cell maturation (Fig I4.1).
small molecules to manipulate specific lineages of the postnatal SVZ and promote cortical repair in the context of chronic hypoxia. The mechanisms involved in cortical repair investigated in the thesis are: neural stem cells and progenitor recruitment and depletion, correct specification and integration of the newborn glutamatergic neurons, and eventually, correct maturation and survival of newborn oligodendrocytes.
The main objective of my thesis was to explore the competency of postnatal neural stem cells to participate to forebrain cortical repair and design new means to encourage this cortical repair in a lineage specific manner. The project had three thematic approaches:
Aim 1 : To investigate the contribution of the dorsal SVZ to the spontaneous cellular repair observed following chronic neonatal hypoxia.
We first investigated if the dorsal microdomain of the postnatal subventricular zone (dorsal SVZ), which continues producing oligodendrocyte and glutamatergic neuron progenitors throughout life, was a source of cell for cortical repair following chronic hypoxia. For this purpose, after validating the model by insuring that apoptosis indeed occurred in the cortex, we fate mapped specifically postnatal dorsal SVZ stem cells after the necessary time for cellular recovery after chronic hypoxia. We also investigated the nature of the cells originating from the dorsal SVZ that migrated in the cortex to conclude on the competency of postnatal dorsal SVZ neural stem cells to roduce cortical neurons expressing layer specific markers.

Aim 2 : To develop a pharmacogenomics approach for identifying small molecules for lineage specific manipulation of postnatal forebrain germinal activity

The second aim of our work consisted in identifying the most efficient small molecule to promote post-hypoxia cortical repair. For this purpose, we performed a pharmacogenomic analysis to highlight small molecules with the potential to trigger oligodendrocyte and glutamatergic neuron fates. Thuis approach was validated by testing small molecules in the neonatal brain, in the adult brain and in the hypoxic brain, thereby showing the amenability of the dorsal SVZ stem cells to pharmacological treatment in different contexts. CHIR99021, a Gsk3β inhibitor, was eventually selected for its efficiency to induce dorsal SVZ cell proliferation, oligodendrocyte and glutamatergic neuron fates, but also for its efficiency using intranasal administration.

Aim 3 : To test small molecules to promote tissue repair following chronic neonatal hypoxia.

The last aim of my thesis was to determine the effect of CHIR99021, on the progeny of dorsal SVZ stem cells in the context of chronic hypoxia. To do this, CHIR99021 was dministrated intranasally following the hypoxic period in the chronic. The effect of the drug on oligodendrocyte maturation, neuronal specification and on the maintenance of the pool of stem cells in the niche were investigated at different timepoints.

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MATERIAL AND METHODS

Unless stated, all materials were purchased from Sigma-Aldrich. All procedures were in accordance and approvals of the UK Home Office Animals Scientific Procedures Act (1986), Ethics Committee of the Veterinary Department of the Canton of Zurich (Approval ID 182/2011). Experiments in France were performed in accordance with European requirements 2010/63/UE and have been approved by the Animal Care and Use Committee CELYNE (APAFIS#187 & 188). Animal procedures were executed in accordance with UK/Swiss/French law, with strict consideration given to the care and use of animals. All mice were bred over wildtype C57/BL6 background for several generations and positive animals for Mash1-EGFP selected at birth under UV light.

Part performed by Kasum Azim Bioinformatics

Whole genome transcriptome datasets of the isolated SVZ microdomains, and regionspecific NSCs and TAPs are described in detail in a recent study that aimed at describing transcriptional regulators acting in SVZ regionalization [1]. Briefly, the rostral periventricular regions of postnatal mice of different ages (P4, P8 and P11) of the Ascl1-EGFPBac transgenic reporter mouse line were carefully microdissected under a fluorescent binocular microscope in RNAse free and sterile conditions. SVZ microdomains isolated were derived from brain coordinates +1 relative to the bregma to 0 relative to the bregma. The Hes5-EGFP reporter mouse line was used in combination with Prominin-1 immunodetection to isolate NSCs from microdissected dorsal and lateral microdomains by fluorescence activated cell sorting. Similarly, the Ascl1-EGFPBac transgenic reporter mouse line was used to isolate the 25% brightest cells, i.e. corresponding to TAPs, from either microdomain. Half a litter of animals were used to pool for each replicate throughout. In the present study, these datasets (recently made publically available from NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) GEO Series accession number GSE60905), were analyzed using previously applied bioinformatics methods, with only minor modifications (Azim et al., 2015). In brief, data were cured (background subtraction, normalization, and summarization) using robust multi-chip analysis (RMA) using the Partek Genomic Suite software package version 6.6 using stringent false discovery rate (FDR) with p-values where necessary in the analysis. All data’s were normalized collectively with datasets from previous studies of isolated NSC, NPs and glia (i.e. GSE60905, GSE9566, GSE18765) for optimal parameters. Partek was used assemble affymetrix data to generate hierarchical clustering and gene lists. GO’s were generated using the latest MGI mouse GO datasets via the Broad Institute (http://www.broadinstitute.org/gsea/index.jsp). The numbers of probe that were differentially expressed across the 10 samples analysed (dNSCs, dTAPs, lNSCs, lTAPs, P4 dorsal SVZ, P4 lateral SVZ, P8 dorsal SVZ, P8 lateral SVZ, P11 dorsal SVZ and P11 lateral SVZ) represented a total of ~37K probesets within the 10% FDR range.
Genego Metacore (https://portal.genego.com/) and GSEA (http://www.broadinstitute.org/gsea/msigdb/index.jsp) were used to filter and select for probes associated as secreted morphogens (tropic factors, growth factors, extracellular signaling molecules, mitogens and secreted inhibitors of signaling pathways). The numbers of morphogen from this filtered list that were significantly altered amounted to 530 probes, representing approximately 330 individual genes.
Identification of spatially enriched signaling ligands regardless of sample type (Fig 1AB) was done by comparing all dorsal versus all lateral samples. This gene list was uploaded onto Genego Metacore and Process Network option selected using the default parameters. Determination of the spatial expression profiles of secreted signaling factors in SVZ microdomains (Fig 1C-H) was performed by comparing datasets using appropriate fold changes and FDR cut-offs (Partek, 1.65 fold change and FDR < 5%). For all analysis, raw expression values are provided and Heatmaps are presented in the manuscript.

Table of contents :

CHAPTER 1 : Dorsal subventricular zone neural stem cells contribute to de novo cortical oligodendrogenesis and neurogenesis following chronic neonatal hypoxia.
SUMMARY – Chapter 1
RESULTS – Chapter 1
DISCUSSION – Chapter 1
Supplementary data – Chapter 1
CHAPTER 2 : Pharmacogenomic identification of small molecules for lineage specific manipulation of subventricular zone germinal activity
SUMMARY – Chapter 2
RESULTS – Chapter 2
DISCUSSION – Chapter 2
Supplementary data – Chapter 2
CHAPTER 3 : Intranasal administration of CHIR99021 promotes cellular repair following neonatal hypoxia
SUMMARY – Chapter 3
RESULTS – Chapter 3
DISCUSSION – Chapter 3
Supplementary data – Chapter 3
GENERAL CONCLUSION & PERSPECTIVES

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