Discussion 215 7 1.1 Interneuron vulnerability We investigated if we could pinpoint interneuron subtype vulnerability. Analysis of genes associated with interneuron subtypes revealed increased ERBB4 expression in 4H cortical neurons both with qPCR (Chapter 2) and bulk mRNA sequencing of mono-culture setups (Chapter 3). ERBB4 expression is related to parvalbumin (PV) interneuron development. Conversely, no changes were detected in RELN (NDNF interneurons) or CNTNAP2 (VIP interneurons) using qPCR, though bulk sequencing on mono-cultures suggested modest alterations (RELN; CNTNAP2). Also in the spheroid models, single-cell RNA sequencing revealed changes in interneuron related genes, specifically downregulation of SST expression in the cell cluster SST-expressing GABAergic neurons on both day 100 and 150 (Chapter 4). This consistent dysregulation supports the hypothesis that specific interneuron subclasses, particularly PV- and SST-interneurons, may be more vulnerable in 4H pathology. However, it remains unclear whether interneurons represent primarily affected cell types or if their dysfunction is a secondary consequence of broader neuronal deficits. Interneurons are known to play critical roles in coordinating cortical circuits, and their impairment may exacerbate functional deficits observed in 4H. Future research should evaluate whether interneurons can be selectively targeted for therapeutic interventions and whether these strategies may ameliorate cellular phenotypes in vitro. 1.2 Sonic hedgehog pathway dysregulation As mentioned our investigations identified ARX downregulation in cerebellar differentiation products, corroborated by qPCR findings in cortical co-cultures. Since ARX is an important regulator of sonic hedgehog (Shh) gradients during development (Cho et al., 2014), we hypothesized that interneuron development is possibly affected through the Shh pathway. But ARX downregulation was not replicated in neuron mono-cultures, possibly as this difference is cell stage-dependent. And, therapeutic targeting of the Shh pathway using SAG failed to restore neuronal function (Chapter 2), Interestingly, ARX is not the only dysregulated gene in 4H that can be linked to the Shh pathway. ITGA11 was significantly upregulated in cerebellar differentiation products of 4H. It was shown in human hepatic stellate cells that ITGA11 can be regulated by the hedgehog signalling pathway, and inhibition of the hedgehog pathway can reduce ITGA11 (Bansal et al., 2017). Also, CHL1, which exhibited significant yet modest downregulation in 4H cortical mono-cultures (Chapter 3), can be linked to Shh. Specifically, since CHL1 is known to interact with Ptch1 during neuronal apoptosis and cerebellar development (Huang et al., 2011). Ptch1 is a receptor of hedgehog and regulates proliferation, neurogenesis and axon guidance
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