Cell-type-specific regulation of APOE and CLU levels in human neurons by the Alzheimer's disease risk gene SORL1

SORL1 is strongly implicated in the pathogenesis of Alzheimer's disease (AD) through human genetic studies that point to an association of reduced SORL1 levels with higher risk for AD. To interrogate the role(s) of SORL1 in human brain cells, SORL1 null iPSCs were generated, followed by differentiation to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 led to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. Intriguingly, SORL1 loss led to a dramatic neuron-specific reduction in APOE and CLU levels, an increase in the accumulation of lipid droplets and altered lipid profiles. Pathway analysis implicated intracellular transport pathways and TGF-ß/SMAD signaling in the function of SORL1 in neurons. Enhancement of retromer-mediated trafficking and autophagy rescued tau phenotypes observed in SORL1 null neurons but did not rescue APOE levels. However, stimulation and inhibition of SMAD signaling in neurons modulated APOE RNA levels in a SORL1-dependent manner. Further, analyses of iPSCs derived from a human aging cohort revealed a neuron-specific linear correlation between SORL1 and APOE RNA and protein levels, a finding validated in human post-mortem brain. These studies provide a mechanistic link between two of the strongest genetic risk factors for AD. Overall design: The CVB (Ventner) iPSC line was CRISPR modified to eliminate expression of the SORL1 gene (SORL1-KO), control lines went through the CRISPR process but resulted in no modification of SORL1 (SORL1-WT). Both SORL1 WT and KO lines were harvested in 3 replicates either as indifferentiated iPSC cells or as differentiated neurons (iN), astrocytes (iAst), microglia (iMGL), or endothelial cells (iEC). Briefly iNs were generated by direct differentiation by expression of neurogenin-2 using a modified version of the process originally described in Zhang et al. (Neuron 78(5), 2013). Astrocytes were generate by expression of SOX9 and NIFB using a modified version of the protocol described in Canals et al. (Nature Methods 15, 2018). Differentiated neurons and astrocytes were both harvested on d21 of differentiation. Microglia were differentiated by first exposing iPSC colonies to hematopoietic differentiation conditions using the STEMdiff Kit from STEMCELL technologies and then exposing the resultant CD34+, CD45+, CD43+ cells to exogenous differentition factors as thoroughly described in Abud et al. (Neuron 94(2), 2017). Differentiated microglia were harvested at d41 of the protocol. Endothelial Like Cells (iECs), were differentiated with minor modification according to the protocols of Lippmann et al., 2012 , Lippmann et al., 2015 , and Park et al., 2019.