Cadherin-13 is a critical regulator of GABAergic modulation in human stem-cell-derived neuronal networks

Abstract

Activity in the healthy brain relies on a concerted interplay of excitation (E) and inhibition (I) via balanced synaptic communication between glutamatergic and GABAergic neurons. A growing number of studies imply that disruption of this E/I balance is a commonality in many brain disorders; however, obtaining mechanistic insight into these disruptions, with translational value for the patient, has typically been hampered by methodological limitations. Cadherin-13 (CDH13) has been associated with autism and attention-deficit/hyperactivity disorder. CDH13 localizes at inhibitory presynapses, specifically of parvalbumin (PV) and somatostatin (SST) expressing GABAergic neurons. However, the mechanism by which CDH13 regulates the function of inhibitory synapses in human neurons remains unknown. Starting from human-induced pluripotent stem cells, we established a robust method to generate a homogenous population of SST and MEF2C (PV-precursor marker protein) expressing GABAergic neurons (iGABA) in vitro, and co-cultured these with glutamatergic neurons at defined E/I ratios on micro-electrode arrays. We identified functional network parameters that are most reliably affected by GABAergic modulation as such, and through alterations of E/I balance by reduced expression of CDH13 in iGABAs. We found that CDH13 deficiency in iGABAs decreased E/I balance by means of increased inhibition. Moreover, CDH13 interacts with Integrin-β1 and Integrin-β3, which play opposite roles in the regulation of inhibitory synaptic strength via this interaction. Taken together, this model allows for standardized investigation of the E/I balance in a human neuronal background and can be deployed to dissect the cell-type-specific contribution of disease genes to the E/I balance.

Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro

“…To investigate if neuronal network activity from hiPSCderived Ngn2-induced excitatory neurons was reproducible, we performed a meta-analysis on MEA data derived from multiple control lines used in our lab (Frega et al, 2019;Klein Gunnewiek et al, 2020;Mossink et al, 2021). The control lines were derived from fibroblast skin biopsies from ten healthy individuals, five males and five females, with a mean age of 33.5 years (Figure 1A), and we extracted 17 parameters in total to describe the neuronal network activity and connectivity (Table S1).…”

A Common CDH13 Variant Is Associated with Low Agreeableness and Neural Responses to Working Memory Tasks in ADHD

“…Therefore, future studies that focus on CDH13 in neurodevelopmental disorders should concentrate on the role of CDH13-mediated neuronal outgrowth in attentional networks, such as frontostriatal circuits and hippocampal regions, in both rodent models and human studies. For the former, a conditional knockout mouse is already under investigation [70]; to address the latter, CDH13 +/− and CDH13 −/− human-induced pluripotent cell lines have been created recently [71] and can be used to investigate the impact of CDH13 on the excitatory-inhibitory balance in neuronal cell models [72].…”

Functional Characterization of Human Pluripotent Stem Cell-Derived Models of the Brain with Microelectrode Arrays

“…A similar protocol utilizing lentiviral overexpression of the mouse transcription factors distal-less homeobox 2 (Dlx2) and achaete-scute family bHLH transcription factor 1 (Ascl1) was used to create pure GABAergic populations [ 133 ] that are functional on MEA after only two weeks of maturation [ 134 ]. Even Ascl1 overexpression alone was shown to induce GABAergic neurons that, under the influence of MEA, produce mean firing rates (MUA) of approximately 5 Hz and 3–4 synchronized bursts per min [ 135 ]. While the bursting can be seen as contradictory to human in vivo data, which suggest that inhibitory neurons rarely burst, it is impossible to say whether the in vivo recordings have truly identified GABAergic populations due to the absence of any molecular analysis [ 21 ].…”

“…While the bursting can be seen as contradictory to human in vivo data, which suggest that inhibitory neurons rarely burst, it is impossible to say whether the in vivo recordings have truly identified GABAergic populations due to the absence of any molecular analysis [ 21 ]. It is also possible that the experimental conditions in the in vitro preparations [ 135 ] encourage bursting. Furthermore, the in vitro and in vivo MEA data should be analyzed for SUA to compare the function of the supposed GABAergic neurons [ 21 , 135 ].…”