A Quantitative LC-MS/MS Method for Distinguishing the Tau Protein Forms Phosphorylated and Nonphosphorylated at Serine-396.

Hyperphosphorylated tau protein is well-known to be involved in the formation of neurofibrillary tangles and the progression of age-related neurodegenerative diseases (tauopathies), including Alzheimer's Disease (AD). Tau protein phosphorylated at serine-396 (pS396-tau) is often linked to disease progression, and we therefore developed an analytical method to measure pS396-tau in cerebrospinal fluid (CSF) in humans and animal models of AD. In the S396-region, multiple phosphorylation sites are present, causing structural complexity and sensitivity challenges for conventional bottom-up mass spectrometry approaches. Here, we present an indirect LC-MS/MS method for quantification of pS396-tau. We take advantage of the reproducible miscleavage caused by S396 being preceded by a lysine (K395) and the proteolytic enzyme trypsin not cleaving when the following amino acid is phosphorylated. Therefore, treatment with trypsin discriminates between the forms of tau with and without phosphorylation at S396 and pS396-tau can be quantified as the difference between total S396-tau and nonphosphorylated S396-tau. To qualify the method, it was successfully applied for quantification of pS396-tau in human CSF from healthy controls and patients with Mild Cognitive Impairment and AD. In addition, the method was applied for rTg4510 mice where a clear dose dependent decrease in pS396-tau was observed in CSF following intravenous administration of a monoclonal antibody (Lu AF87908, hC10.2) targeting the tau epitope containing pS396. Finally, a formal validation of the method was conducted. In conclusion, this sensitive LC-MS/MS-based method for measurement of pS396-tau in CSF allows for quantitative translational biomarker applications for tauopathies including investigations of potential drug induced effects.

Epithelial Barrier Integrity Profiling: Combined Approach Using Cellular Junctional Complex Imaging and Transepithelial Electrical Resistance.

A core aspect of epithelial cell function is barrier integrity. A loss of barrier integrity is a feature of a number of respiratory diseases, including asthma, allergic rhinitis, and chronic obstructive pulmonary disease. Restoration of barrier integrity is a target for respiratory disease drug discovery. Traditional methods for assessing barrier integrity have their limitations. Transepithelial electrical resistance (TEER) and dextran permeability methods can give poor in vitro assay robustness. Traditional junctional complex imaging approaches are labor-intensive and tend to be qualitative but not quantitative. To provide a robust and quantitative assessment of barrier integrity, high-content imaging of junctional complexes was combined with TEER. A scalable immunofluorescent high-content imaging technique, with automated quantification of junctional complex proteins zonula occludens-1 and occludin, was established in 3D pseudostratified primary human bronchial epithelial cells cultured at an air-liquid interface. Ionic permeability was measured using TEER on the same culture wells.The improvements to current technologies include the design of a novel 24-well holder to enable scalable in situ confocal cell imaging without Transwell membrane excision, the development of image analysis pipelines to quantify in-focus junctional complex structures in each plane of a Z stack, and the enhancement of the TEER data analysis process to enable statistical evaluation of treatment effects on barrier integrity. This novel approach was validated by demonstrating measurable changes in barrier integrity in cells grown under conditions known to perturb epithelial cell function.

Single-Step, High-Efficiency CRISPR-Cas9 Genome Editing in Primary Human Disease-Derived Fibroblasts.

Genome editing is a tool that has many applications, including the validation of potential drug targets. However, performing genome editing in low-passage primary human cells with the greatest physiological relevance is notoriously difficult. High editing efficiency is desired because it enables gene knockouts (KO) to be generated in bulk cellular populations and circumvents the problem of having to generate clonal cell isolates. Here, we describe a single-step workflow enabling >90% KO generation in primary human lung fibroblasts via CRISPR ribonucleoprotein delivery in the absence of antibiotic selection or clonal expansion. As proof of concept, we edited two SMAD family members and demonstrated that in response to transforming growth factor beta, SMAD3, but not SMAD2, is critical for deposition of type I collagen in the fibrotic response. The optimization of this workflow can be readily transferred to other primary cell types.