The barrier function of the human epidermis is constantly challenged by environmental osmotic fluctuations. Hypotonic stress triggers cell swelling, which is counteracted by a compensatory mechanism called regulatory volume decrease (RVD) involving volume‐regulated anion channels (VRACs). Recently, it was discovered that VRACs are composed of LRRC8 heteromers and that LRRC8A functions as the essential VRAC subunit in various mammalian cell types; however, the molecular identity of VRACs in the human epidermis remains to be determined. Here, we investigated the expression of LRRC8A and its role in hypotonic stress response of human keratinocytes. Immunohistological staining showed that LRRC8A is preferentially localized in basal and suprabasal epidermal layers. RNA sequencing revealed that LRRC8A is the most abundant subunit within the LRRC8 gene family in HaCaT cells as well as in primary normal human epidermal keratinocytes (NHEKs). To determine the contribution of LRRC8A to hypotonic stress response, we generated HaCaT‐ and NHEK‐LRRC8A knockout cells by using CRISPR‐Cas9. I− influx assays using halide‐sensitive YFP showed that LRRC8A is crucially important for mediating VRAC activity in HaCaTs and NHEKs. Moreover, cell volume measurements using calcein‐AM dye further revealed that LRRC8A also substantially contributes to RVD. In summary, our study provides new insights into hypotonic stress response and suggests an important role of LRRC8A as VRAC component in human keratinocytes.

Alzheimer's disease (AD) pathology precedes the onset of clinical symptoms by several decades. Thus, biomarkers are required to identify prodromal disease stages to allow for the early and effective treatment. The methoxy-X04-derivative BSC4090 is a fluorescent ligand which was designed to target neurofibrillary tangles in AD. BSC4090 staining was previously detected in post-mortem brains and olfactory mucosa derived from AD patients. We tested BSC4090 as a potential diagnostic marker of prodromal and early AD using olfactory mucosa biopsies from 12 individuals with AD, 13 with mild cognitive impairment (MCI), and 10 cognitively normal (CN) controls. Receiver-operating curve analysis revealed areas under the curve of 0.78 for AD versus CN and of 0.86 for MCI due to AD versus MCI of other causes. BSC4090 labeling correlated significantly with cerebrospinal fluid levels of tau protein phosphorylated at T181. Using NMR spectroscopy, we find that BSC4090 binds to fibrillar and pre-fibrillar but not to monomeric tau. Thus, BSC4090 may be an interesting candidate to detect AD at the early disease stages.

Cell adhesion molecules are crucial for a variety of biological processes, including wound healing, barrier formation and tissue homeostasis. One of them is E-cadherin which is generally found at adherent junctions between epithelial cells. To identify this molecule on the surface of cells, E-cadherin mimetic peptides with a critical amino acid sequence of HAV (histidine-alanine-valine) were synthesized and attached to solid-supported membranes covering colloidal probes. Two different functionalization strategies were established, one based on the complexation of DOGS-NTA(Ni) with a polyhistidine-tagged HAV-peptide and the other one relying on the formation of a HAV-lipopeptide using in situ maleimide-thiol coupling. Binding studies were performed to verify the ability of the peptides to attach to the membrane surface. Compared to the non-covalent attachment via the His-tag, we achieved a higher yield by lipopeptide formation. Colloidal probes functionalized with HAV-peptides were employed to measure the presence of E-cadherins on living cells either using video particle tracking or force spectroscopy. Here, human HaCaT cells were examined confirming the specific interaction of the HAV-peptide with the Ecadherin of the cells. Statistical methods were also used to determine the number of single-bond ruptures and the force of a single bond. These findings may be essential for the development of novel biosynthetic materials given their potential to become increasingly relevant in medical applications.

Filaggrin (FLG) loss-of-function mutations and the T helper 2 (Th2) dominated cytokine milieu are assumed to cause an impaired skin barrier function in atopic dermatitis (AD), but this presumed mechanism is still largely hypothetical. Previous studies have used in vitro skin equivalents to provide experimental evidence for the role of FLG deficiency but different experimental setups and incomplete knockdown approaches make these data difficult to compare and interpret. Using 3D epidermal equivalents, we here addressed the question if FLG deficiency alters skin barrier function. We excluded interplay of FLG mutations with other AD-typical concomitant factors like inflammation or altered microbiome. We therefore used keratinocytes of ichthyosis vulgaris patients that innately carry homozygous FLG null mutations. This approach uses genetically defined cells without detectable filaggrin protein and avoids potential off-target effects of knockdown approaches. FLG did not alter constitutive or Th2-cytokine dependent expression of differentiation-associated proteins. We observed a decrease of tight junction protein expression, which, however, did not lead to alteration of the outside-in nor did it affect inside-out stratum corneum barrier as measured by permeability for low molecular weight tracers (Lucifer Yellow or biotin-SH). Although these findings do not completely rule out alterations in epidermal permeability for molecules with other biophysical properties, our study contradicts previous work that suggests an increased permeability for low molecular weight polar solutes in FLG deficient epidermis.