The role of the CRH/CRHR1 system in glial cells following acute brain injury

Ries, Clemens (2024): The role of the CRH/CRHR1 system in glial cells following acute brain injury. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)

Abstract

This cumulative-style doctoral thesis consists of two studies revolving around the corticotropin-releasing hormone (CRH), its role in oligodendrogenic processes following injury as well as the in-depth characterization of mouse models for its analysis. We identified a subpopulation of oligodendrocyte progenitor cells (OPCs) in which CRH expression is triggered upon acute injury by using different CRH reporter models, direct anti-CRH staining and Crh mRNA visualization. This CRH-expressing subpopulation of OPCs showed an OPC-like injury response with inward migration, strong proliferation and later differentiation. Although the general injury reaction of CRH+ OPCs was comparable to the whole population of OPCs, their dynamics differed, supposedly caused by their high maturation rate of 80%. CRH expression was further found to happen at very early stages, starting within the first 12h following injury, therefore, preceding proliferation. Subsequently, expression of CRH persisted only within the first 3 days post injury (dpi). Furthermore, CRHR1, the high affinity receptor of CRH, was identified on OPCs and astrocytes surrounding the injury site, serving as potential targets of injury-triggered CRH expression and release. Using different gain- and loss-of-function approaches we demonstrated that CRH modulates astrocytic activation stages in a CRHR1-dependent manner, as elevated GFAP levels were only observed following CRH injection when CRHR1 was present. Furthermore, CRH system downregulation by global CRHR1 or OPC-specific CRH inactivation led to a reduced number of OPCs at 7 dpi and an increased number of OLs, that was later explained by an increased generation of new OLs using a label retaining experiment. Therefore, CRH released from OPCs apparently triggers a stalling mechanism of OPC differentiation which is mediated by CRHR1-expressing OPCs and astrocytes. In sum this study presents a novel neuropeptide system modulating OPC differentiation processes, which could serve as therapeutic target to enhance OL regeneration following acute injury. This study was complemented by a much-needed characterization of a newly available CRH-FlpO reporter line. In this study, the CRH-FlpO line was compared to the widely used CRH-Cre line, which is considered as the gold standard for transgenic CRH reporting. By using 9 different reporter lines we were able to show that FlpO-dependent reporting in CRH-FlpO::Ai65F mice, although replicating endogenous CRH expression patterns, is inferior in its capacity to report the whole population of CRH-expressing cells. By using Cre and FlpO in combination with different reporters, we demonstrated that overall recombination efficacy of FlpO is lower than that of Cre. Additionally, we determined that an elevation in CRH expression by applying an acute stress model increases the number of reported cells. Therefore, FlpO dependent recombination and reporting is highly dependent on the overall strength but also context-dependent activity of the driving promoter. This has consequences for all studies using mouse lines harboring FlpO for genetic recombination: i) When comparing Cre- and FlpO-driven reporting the strength of the driving promoter has to be taken into account, ii) when using intersectional approaches to label specific subpopulations the efficacy of reporting is determined by the recombination efficiency of FlpO and iii) when using FlpO for overexpression or knock-out of a certain marker an in-depth validation of their efficacy in the specific population is necessary. In sum, our results show that a careful and comprehensive analysis of FlpO-driven mouse lines is mandatory and should pay particular attention to the expression strength of the driving promoter. Results connected to the characterization of FlpO were essential when interpreting the data related to the identification of CRH expression in OPCs. Because of the limited accessibility of CRH and its receptor CRHR1 by antibody-mediated approaches, the study relied heavily on transgenic mouse models, using both Cre and FlpO. Especially, differences in the number of reported cells between different mouse lines could be explained by these results which made a reasonable evaluation of gathered data possible. In sum, this thesis presents a thorough evaluation study of FlpO driven recombination efficacy whose results laid the ground for the identification and characterization of a novel OPC-derived neuropeptide system contributing to OLC differentiation processes.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: CRH, Injury, OPCs, oligodendrocytes
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Graduate School of Systemic Neurosciences (GSN)
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 16. Mai 2024
• 1. Bericht­erstatter:in: Deussing, Jan