Reepithelialization in focus: Non-invasive monitoring of epidermal wound healing in vitro.

Up to today, in vivo studies are the gold standard for testing of new therapeutics for cutaneous wound healing. Alternative in vitro studies are mostly limited to two-dimensional cell cultures and thus only poorly reflect the complex physiological wound situation. Here we present a new three-dimensional wound model based on a reconstructed human epidermis (RHE). We introduce impedance spectroscopy as a time-resolved test method to determine the efficacy of wound healing non-destructively by focusing on the barrier function of the RHE as a main feature of intact skin. We assessed the skin barrier quantitatively and qualitatively by calculating the transepithelial electrical resistance (TEER), by fitting an equivalent circuit and by analyzing the single characteristic frequency. Upon wounding using a 2 mm biopsy punch, the impedance dropped significantly to 3.5% of the initial value. Impedance spectroscopy thereby proved to be a sensitive tool to distinguish between wounds of different sizes. The glucose and lactate concentration in the medium revealed an acute stress reaction of the wounded RHE (wRHE) in the first days after wounding. During monitoring of reepithelialization over fourteen days, the barrier fully recovered. Microscopy and histology images correlate well with these findings, revealing an active wound closure mostly completed by day seven after wounding. These wounded epidermal models can now be applied in therapeutic screenings and with the help of rapid screening by impedance spectroscopy, expensive and time-consuming imaging and histological methods as well as the use of animal models can be reduced.

Disruption of a Structurally Important Extracellular Element in the Glycine Receptor Leads to Decreased Synaptic Integration and Signaling Resulting in Severe Startle Disease.

Functional impairments or trafficking defects of inhibitory glycine receptors (GlyRs) have been linked to human hyperekplexia/startle disease and autism spectrum disorders. We found that a lack of synaptic integration of GlyRs, together with disrupted receptor function, is responsible for a lethal startle phenotype in a novel spontaneous mouse mutant shaky, caused by a missense mutation, Q177K, located in the extracellular ß8-ß9 loop of the GlyR α1 subunit. Recently, structural data provided evidence that the flexibility of the ß8-ß9 loop is crucial for conformational transitions during opening and closing of the ion channel and represents a novel allosteric binding site in Cys-loop receptors. We identified the underlying neuropathological mechanisms in male and female shaky mice through a combination of protein biochemistry, immunocytochemistry, and both in vivo and in vitro electrophysiology. Increased expression of the mutant GlyR α1Q177K subunit in vivo was not sufficient to compensate for a decrease in synaptic integration of α1Q177Kß GlyRs. The remaining synaptic heteromeric α1Q177Kß GlyRs had decreased current amplitudes with significantly faster decay times. This functional disruption reveals an important role for the GlyR α1 subunit ß8-ß9 loop in initiating rearrangements within the extracellular-transmembrane GlyR interface and that this structural element is vital for inhibitory GlyR function, signaling, and synaptic clustering.SIGNIFICANCE STATEMENT GlyR dysfunction underlies neuromotor deficits in startle disease and autism spectrum disorders. We describe an extracellular GlyR α1 subunit mutation (Q177K) in a novel mouse startle disease mutant shaky Structural data suggest that during signal transduction, large transitions of the ß8-ß9 loop occur in response to neurotransmitter binding. Disruption of the ß8-ß9 loop by the Q177K mutation results in a disruption of hydrogen bonds between Q177 and the ligand-binding residue R65. Functionally, the Q177K change resulted in decreased current amplitudes, altered desensitization decay time constants, and reduced GlyR clustering and synaptic strength. The GlyR ß8-ß9 loop is therefore an essential regulator of conformational rearrangements during ion channel opening and closing.