Single-cell RNA sequencing reveals 2D cytokine assay can model atopic dermatitis more accurately than immune-competent 3D setup.

Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL-4 and IL-13 induce AD-like features in keratinocytes in vitro. However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure is superior to model AD compared to simple 2D cell culture with cytokines. For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single-cell RNA sequencing data (scRNA-seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL-4 and IL-13 induced the closest in vivo-like AD phenotype which was observed in the scRNA-seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM-associated genes such as POSTN. While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo. Taken together, our comprehensive study shows that the simple model using IL-4 or IL-13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro, which may aid the discovery of novel treatment options.

In vitro models for investigating itch.

Itch (pruritus) is a sensation that drives a desire to scratch, a behavior observed in many animals. Although generally short-lasting and not causing harm, there are several pathological conditions where chronic itch is a hallmark symptom and in which prolonged scratching can induce damage. Finding medications to counteract the sensation of chronic itch has proven difficult due to the molecular complexity that involves a multitude of triggers, receptors and signaling pathways between skin, immune and nerve cells. While much has been learned about pruritus from in vivo animal models, they have limitations that corroborate the necessity for a transition to more human disease-like models. Also, reducing animal use should be encouraged in research. However, conducting human in vivo experiments can also be ethically challenging. Thus, there is a clear need for surrogate models to be used in pre-clinical investigation of the mechanisms of itch. Most in vitro models used for itch research focus on the use of known pruritogens. For this, sensory neurons and different types of skin and/or immune cells are stimulated in 2D or 3D co-culture, and factors such as neurotransmitter or cytokine release can be measured. There are however limitations of such simplistic in vitro models. For example, not all naturally occurring cell types are present and there is also no connection to the itch-sensing organ, the central nervous system (CNS). Nevertheless, in vitro models offer a chance to investigate otherwise inaccessible specific cell-cell interactions and molecular pathways. In recent years, stem cell-based approaches and human primary cells have emerged as viable alternatives to standard cell lines or animal tissue. As in vitro models have increased in their complexity, further opportunities for more elaborated means of investigating itch have been developed. In this review, we introduce the latest concepts of itch and discuss the advantages and limitations of current in vitro models, which provide valuable contributions to pruritus research and might help to meet the unmet clinical need for more refined anti-pruritic substances.

Hyperactivity is a Core Endophenotype of Elevated Neuregulin-1 Signaling in Embryonic Glutamatergic Networks.

The neuregulin 1 (NRG1) ErbB4 module is at the core of an "at risk" signaling pathway in schizophrenia. Several human studies suggest hyperstimulation of NRG1-ErbB4 signaling as a plausible pathomechanism; however, little is known about the significance of stage-, brain area-, or neural cell type-specific NRG1-ErbB4 hyperactivity for disease-relevant brain endophenotypes. To address these spatiotemporal aspects, we generated transgenic mice for Cre recombinase-mediated overexpression of cystein-rich domain (CRD) NRG1, the most prominent NRG1 isoform in the brain. A comparison of "brain-wide" vs cell type-specific CRD-NRG1 overexpressing mice revealed that pathogenic CRD-NRG1 signals for ventricular enlargement and neuroinflammation originate outside glutamatergic neurons and suggests a subcortical function of CRD-NRG1 in the control of body weight. Embryonic onset of CRD-NRG1 in glutamatergic cortical networks resulted in reduced inhibitory neurotransmission and locomotor hyperactivity. Our findings identify ventricular enlargement and locomotor hyperactivity, 2 main endophenotypes of schizophrenia, as specific consequences of spatiotemporally distinct expression profiles of hyperactivated CRD-NRG1 signaling.

NeuroD2 controls inhibitory circuit formation in the molecular layer of the cerebellum.

The cerebellar cortex is involved in the control of diverse motor and non-motor functions. Its principal circuit elements are the Purkinje cells that integrate incoming excitatory and local inhibitory inputs and provide the sole output of the cerebellar cortex. However, the transcriptional control of circuit assembly in the cerebellar cortex is not well understood. Here, we show that NeuroD2, a neuronal basic helix-loop-helix (bHLH) transcription factor, promotes the postnatal survival of both granule cells and molecular layer interneurons (basket and stellate cells). However, while NeuroD2 is not essential for the integration of surviving granule cells into the excitatory circuit, it is required for the terminal differentiation of basket cells. Axons of surviving NeuroD2-deficient basket cells follow irregular trajectories and their inhibitory terminals are virtually absent from Purkinje cells in Neurod2 mutants. As a result inhibitory, but not excitatory, input to Purkinje cells is strongly reduced in the absence of NeuroD2. Together, we conclude that NeuroD2 is necessary to instruct a terminal differentiation program in basket cells that regulates targeted axon growth and inhibitory synapse formation. An imbalance of excitation and inhibition in the cerebellar cortex affecting Purkinje cell output may underlay impaired adaptive motor learning observed in Neurod2 mutants.