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

Abstract

Activity in the healthy brain relies on 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 human patient, has typically been hampered by methodological limitations.Cadherin-13 (CDH13) has strongly been associated to attention-deficit/hyperactivity disorder and comorbid disorders such as autism and schizophrenia. CDH13 localises 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 PV 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 CDH13deficiency 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. IntroductionNeuronal network activity is controlled by a tightly regulated interplay between excitation (E) and inhibition (I). In the healthy brain, this interplay maintains a certain E/I ratio via balanced synaptic communication between glutamatergic and GABAergic neurons 1, 2 , resulting in the so called 'E/I balance'. A growing number of studies imply that the E/I balance is disrupted in many neurodevelopmental disorders (NDDs) 3, 4 , including monogenic disorders, where the causative mutations are typically related to altered neuronal excitability and/or synaptic communication 5-7 , as well as polygenic disorders, such as autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD) and schizophrenia 4,8 . Mutations in Cadherin-13 (CDH13, also known as T-Cadherin or H-Cadherin) 9 have been associated with ADHD [10][11][12] and comorbid disorders such as ASD, schizophrenia, alcohol dependence and violent behaviour [13][14][15][16] . CDH13 is a special member of the cadherin superfamily since it lacks a transmembrane-and intracellular domain, and in contrast to other Cadherins, is anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor 17,18 . Because of this relatively weak connection to the outer membrane 19 , ...

Loss-of-function variants in the schizophrenia risk gene SETD1A alter neuronal network activity in human neurons through cAMP/PKA pathway

“…We evaluated whether LoF of SETD1A gene results in neuronal network impairment in vitro. To this end, we generated composite networks of glutamatergic (~75%) and GABAergic (~25%) neurons (excitatory/inhibitory cultures, E/I cultures) comprised of either control or SETD1A +/− hiPSCs, by forced expression of the transcription factor Ngn2 or Ascl1, respectively, as recently described ( Figure 1d ) 14 . We did not detect any significant differences in the percentage of glutamatergic or GABAergic neurons between control and SETD1A +/− networks at days in vitro (DIV) 49 ( Figure 1e, S2a ).…”

“…We did not detect any significant differences in the percentage of glutamatergic or GABAergic neurons between control and SETD1A +/− networks at days in vitro (DIV) 49 ( Figure 1e, S2a ). Moreover, following acute treatment with 100 μM Picrotoxin (PTX) at DIV49 on micro-electrode array (MEA), both mean firing rate and network burst duration went up for both control and SETD1A +/− networks ( Figure S2b-c ), indicating that at DIV49 GABAergic neurons exhibit robust inhibitory control 14 .…”

“…The neuronal differentiation protocol used in this article was previously described 14 . Glutamatergic neurons were either cultured alone or in coculture with GABAergic neurons.…”

“…To investigate the role of SETD1A in neuronal network development and synaptic organization, we generated an isogenic human induced pluripotent stem cell (hiPSC) line with SETD1A haploinsufficiency through CRISPR/Cas9. Subsequently, hiPSCs were differentiated into homogenous populations of glutamatergic and GABAergic neurons 14 . In in vitro cultures containing defined compositions of glutamatergic and GABAergic neurons, we comprehensively analyzed molecular, structural and functional neuronal properties from single cells and neuronal networks during development.…”

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

“…To investigate if network activity from hiPSC-derived Ngn2 -induced excitatory neurons are comparable, we performed a meta-analysis on MEA data derived from multiple control lines used in our lab (Frega et al 2019, Gunnewiek et al 2020, Mossink et al 2020). We generated hiPSCs from fibroblast skin biopsies from ten individuals, five males and five females, with a mean age of 33.5 years ( Fig.…”