Astrocytic Sonic Hedgehog signalling in development and disease

Sonic hedgehog (Shh) signalling is a critical pathway in embryonic development, with known roles in cell differentiation, tissue patterning, and the establishment of body axes (Rowitch et al., 1999; Echelard et al., 1993; Ericson et al., 1997; Jessel and Lumsden, 1997). Beyond these canonical roles, recent research has revealed a broader spectrum of Shh functions, encompassing both pre- and post-natal processes in development and disease (Briscoe and Therond, 2013). Notably, research from the Rowitch Laboratory and other have indicated Shh signalling as a potent neuroprotective mechanism, demonstrating significant efficacy in models of neonatal stroke (Nguyen et al., 2021), hypoxic injury (Heine et al., 2011), and neurodegenerative disease (Li et al., 2020). In these settings, Shh signalling has been shown to mitigate neuronal damage, enhance cell survival, and support tissue repair, highlighting its potential as a therapeutic target for conditions marked by neural injury and degeneration. Shh signalling remains active in the postnatal brain, where it regulates intercellular communication among neurons, astrocytes, and microglia, and defines the specialised characteristics of mature glial cells (Xie et al., 2022; Farmer et al., 2016; Hill et al., 2019). This adaptation ensures glia that are finely tuned to the specific demands of the neural microenvironment. Indeed, Shh signalling is active only in distinct subpopulations of astrocytes, indicating that Shh may functionally specialise these cells (Garcia et al., 2010). This specialisation may underlie the diverse roles that astrocytes perform, including synaptic pruning, modulation of inflammatory responses, and the maintenance of overall brain homeostasis (Ben Haim and Rowitch, 2017; Allen, 2014; Allen and Barres, 2009). However, the role of Shh signalling in astrocytes exposed to hypoxia-ischemia is unclear. At a basic level, astrocytes are known to respond to and, in pathology, be sources of Shh (Garcia et al., 2010; Alvarez et al., 2011). However, how Shh signalling contributes to the diversification of astrocyte populations, enhances their resilience, and drives their interactions with other cell types in the neural environment remains inadequately defined. These knowledge gaps critically limit our understanding of how Shh signalling might be leveraged to modulate astrocytic functions for therapeutic benefits. This thesis aims to address these omissions through a combination of in vivo and in vitro approaches, including both wild-type and genetically modified rodent models, alongside human-derived cerebral organoids. This research seeks to elucidate the mechanisms by which Shh signalling instructs astrocyte function, heterogeneity, and their contribution to the broader neural environment. My overarching hypothesis is Shh signalling diversifies astrocyte populations to confer resilience and homeostasis and elicit secondary astrocyte-cell interactions that shape the neural environment. To support this hypothesis, I have established the following specific aims and present the corresponding results. Aim 1: Elucidate the neuroprotective role of Shh in the earliest stages of neonatal hypoxic-ischemic injury. I demonstrated that a single 50 mg/kg dose of SAG post-injury preserves brain volume, reduces cerebral oedema, and minimises blood-brain barrier (BBB) leakage within 24-hrs. Single-nuclei transcriptomics revealed that SAG treatment diversifies astrocytes and preserves a healthy, homeostatic Ast-H population, distinct from the reactive-like Ast-D population in saline-treated controls. Among the factors secreted by Ast-D reactive astrocytes, I identified Sema3c as a hypoxia-induced, Shh-responsive, anti-angiogenic factor that interacts with endothelial cells (ECs) via the Nrp1 receptor. Sema3c is pathologically upregulated in perivascular astrocytes in saline-treated animals and promotes endothelial cell destabilisation, likely contributing to the compromised BBB, secondary neuroinflammation, and secondary cortical injury observed in these animals. Aim 2: Dissect the signalling interactions between deep-layer Shh-positive neurons, Gli1-positive astrocytes, and neighbouring microglia in the post-natal cortex. Shh signalling is known to promote synaptic pruning, but the precise intercellular interactions involved in this process remain incompletely understood. With colleagues, I identified a synaptic phenotype in Il33-/- animals that mirrors the synaptic abundance seen in mutants with impaired astrocytic Shh signalling. Specifically, I show an increase in pre-synaptic puncta in layer V neurons following Il-33 loss, which I attribute to disrupted synapse engulfment by microglia rather than astrocytes. These findings, in concert with existing literature, suggest a sequential trilineage interaction where Shh from neurons signals to neighbouring astrocytes, prompting them to release Il-33, which then binds to its receptor on microglia to facilitate local synapse engulfment of layer V neurons. This tri-lineage model represents a novel mechanism for synapse pruning in cortical layer V. Aim 3: Optimise a human-based in vitro organoid system to model astrocytic Shh signalling in neurodevelopment and disease. I successfully cultured human cerebral organoids (COs) that recapitulate previously published features of cortical development, incorporating key neuroepithelial-derived cell types, including progenitor cells, excitatory neurons, and astrocytes. I hypothesised that COs with spontaneous abundance of choroid plexus (CP) formation may exhibit incorrect dorsal-ventral polarity. To investigate this, I characterised the spatiotemporal expression of telencephalic inductive signals in COs and examined regionalisation through the expression of their downstream transcription factors (TFs). This analysis led to the establishment of a set of standards to reliably assess CO uniformity prior to therapeutic modelling applications. Subsequently, I demonstrated that apical astroglial progenitor cells are enriched for the Shh-responsive gene KCNJ10 (Kir4.1), with overlapping proteomic expression of Shh and Kir4.1 observed near the ventricular regions. Using EdU and Ki67 staining, I elucidated a role for Kir4.1 in regulating astroglial progenitor cell proliferation and cell cycle dynamics. These findings provide further evidence of how Shh signalling dictates the molecular profile of astroglial lineages, resulting in specific functional outcomes. Conclusion: This thesis explores the functional heterogeneity of astrocytic Shh signalling, providing two distinct examples of Shh-driven astrocyte diversity with significant functional outcomes. In both the perinatal injury and postnatal physiological models, Shh signalling in astrocytes regulated the production of secreted proteins that influenced downstream cellular pathways. In the perinatal injury model, Shh activity upregulated astrocytic Sema3c, which interacted with endothelial cells, while in the postnatal physiological model, Shh signalling induced the upregulation of astrocytic Il-33, which interacted with microglial cells. These findings suggest that Shh signalling not only diversifies astrocyte populations but also modulates their secretory profiles, driving critical functional consequences in the neural environment.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Wilson, Aimee

Advisor dc:contributor.advisor

• Rowitch, David

• Astrocytes
• Heterogeneity
• Sonic hedgehog
• Smoothened agonist
• Cortex
• Stroke
• Semaphorin-3C
• Blood-brain-barrier
• Interleukin-33
• Synapse
• Cerebral organoid
• KCNJ10
• Apical radial glia
• Outer radial glia
• Cell-cycle

Licence

• https://www.repository.cam.ac.uk/bitstreams/1d32e104-3e5e-438d-a308-d05d8ed4c9e7/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.118864

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/385106