A luminescent reporter assay to quantify SORL1 ectodomain shedding and retromer-dependent endosome recycling activity.

Growing evidence suggests that defects in endosomal recycling are a causal mechanism for Alzheimer's disease (AD). Sortilin-like receptor 1 (SORL1) is an endosomal sorting receptor that acts together with the retromer complex to facilitate shuttling of cargo from endosomes back to the trans-Golgi network or to the cell surface. Accumulating data indicate that SORL1 dysfunction contributes to AD pathogenesis. SORL1 is trafficked from the endosome to the cell surface in a retromer-dependent process, where it undergoes enzymatic cleavage, resulting in shedding of the SORL1 ectodomain (also known as soluble SORL1). We capitalized on this physiological process to develop and validate a cell-based luminescent reporter assay incorporating enhanced Gaussia luciferase (eGLuc) to quantify soluble SORL1 in the conditioned media as a marker of endosomal recycling function. The shedding of eGLuc-SORL1 provided a reliable luminescent readout correlating with cellular SORL1 expression under conditions of stable and transient transfection in mammalian cell cultures. Using this system, we demonstrated a robust dependence of SORL1 shedding on retromer levels. Pharmacological treatments and manipulations that either inhibited or enhanced retromer activity produced corresponding changes in eGLuc-SORL1 shedding. Furthermore, the assay demonstrated a reduction in SORL1 shedding in cells expressing pathogenic variants associated with AD, supporting its application in evaluating variant pathogenicity. Given its simplicity and cost-effectiveness, this assay is well suited for high-throughput screening of small-molecule drug candidates that modulate SORL1 trafficking and endosomal recycling. In a broader context, it provides a valuable tool for investigating the biological significance of AD-associated SORL1 variants.