Elucidating a microgliaassociated role for SORLA in modulating AD pathogenesis - PROJECT SUMMARY Mutations in SORLA (encoded by SORL1) identified through GWAS and whole exome sequencing analysis have been linked to increased Alzheimer’s disease (AD) risk. Although a neuronal role for SORLA in suppressing amyloidogenic APP processing and consequent Aβ generation has been established, SORLA expression is ~8-fold higher in human microglia compared to neurons, thus implicating a microglial role for SORLA in AD pathogenesis. So far, functional roles for SORLA in microglia have not yet been described. Here, we used CRISPR/Cas9 editing methods to integrate AD-associated A528T and R744X mutations into the SORL1 (encoding SORLA) locus in human H9 embryonic stem cells, which were subsequently differentiated into human microglia-like (hMGL) cells. Comparing transcriptomic profiles between wildtype and AD-associated SORL1 (A528T, R744X) and TREM2 (R47H) mutant hMGLs reveals pathogenic microglia gene signatures such as induced APOE expression previously described in AD mouse models and human AD brain. Our results also show that cultured SORL1R744X and TREM2R47H hMGLs feature defects in Aβ uptake in an APOE-dependent manner, along with impaired Aβ clearance and plaque association in mouse brain xenotransplants by microdialysis/histology. These results provide pioneering evidence that SORLA dysfunction can confer pathogenic expression signatures and impair microglial function. We hypothesize that microglial dysfunction will vary according to domain-specific mutations in the SORLA extracellular region, and that early and late onset SORLA mutations may show differential effects on microglia dysregulation and microglia/neuron interaction. Using our gene editing/human microglial modeling and analysis platform, we will expand our SORLA mutant embryonic stem cell (ESC) panel to include representative early and late AD onset mutations within each of the functional domains in the SORLA extracellular region. We will characterize gene expression profiles of wildtype (WT) and SORLA mutant ESC-derived microglia (“xhMGs”) in vivo by RNAseq/proteomic analysis, as well as functional aspects of microglial function (Aβ uptake, cytokine profiles) in xhMGs xenotransplanted in AD mouse brain. As our results indicate that SORLA mutations such as R744X and A528T upregulate APOE which may trigger certain aspects of microglia function, we will also test whether APOE and other potential drivers of microglia dysfunction epistatically mediate downstream pathogenic behavior in SORLA mutant microglia xenotransplants in AD mouse brain. We will also explore cellular mechanisms associated with various SORLA mutations that drive cellular dysfunction in ESC-derived microglia and neurons; to this end, we will investigate how SORLA mutations in human microglia (xhMGs) interact with neurons in mouse AD brain xenotransplants, as well as WT and SORLA mutant hMGLs/neurons in co-culture. These results will provide us with mechanistic insight into how various mutations can trigger microglia dysfunction, and potentially describe how aspects of microglial and neuronal-related SORLA pathways are affected to alter age-related onset in AD pathogenesis.
