Human APOER2 Isoforms Have Differential Cleavage Events and Synaptic Properties

Abstract

Human apolipoprotein E receptor 2 (APOER2) is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail. APOER2-ECD contains several ligand-binding domains (LBDs) that are organized into exons with aligning phase junctions, which allows for in-frame exon cassette splicing events. We have identified 25 human APOER2 isoforms from cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events within the N-terminal LBD regions compared with six identified in the heart. APOER2 undergoes proteolytic cleavage in response to ligand binding that releases a C-terminal fragment (CTF) and transcriptionally active intracellular domain (ICD). We tested whether the diversity of human brain-specific APOER2 variants affects APOER2 cleavage. We found isoforms with differing numbers of ligand-binding repeats generated different amounts of CTFs compared with full-length APOER2 (APOER2-FL). Specifically, APOER2 isoforms lacking exons 5-8 (Dex5-8) and lacking exons 4-6 (Dex4-6) generated the highest and lowest amounts of CTF generation, respectively, in response to APOE peptide compared with APOER2-FL. The differential CTF generation of Dex5-8 and Dex4-6 coincides with the proteolytic release of the ICD, which mediates transcriptional activation facilitated by the Mint1 adaptor protein. Functionally, we demonstrated loss of mouse Apoer2 decreased miniature event frequency in excitatory synapses, which may be because of a decrease in the total number of synapses and/or VAMP2 positive neurons. Lentiviral infection with human APOER2-FL or Dex4-6 isoform in Apoer2 knockout neurons restored the miniature event frequency but not Dex5-8 isoform. These results suggest that human APOER2 isoforms have differential cleavage events and synaptic properties.

Low-Density Lipoprotein Receptor-Related Protein 8 at the Crossroad between Cancer and Neurodegeneration

“…In this context, Wasser et al, have reported an alternative LRP8 splice variant lacking the O-linked glycosylation region that is cleaved more efficiently than the full-length counterparts [ 130 ]. More recently, it has been shown that LRP8 isoforms with differing numbers of ligand-binding repeats to generate different amounts of CTFs compared with full-length LRP8 [ 135 ].…”

Heterogeneity of novel APOER2 isoforms specific to Alzheimer’s disease impact cellular and synaptic states

“…Since APOER2 isoforms have been shown to modify synaptic function and synapse number (Beffert et al, 2005; Omuro et al, 2022), we performed immunofluorescence labeling using antibodies against the presynaptic protein synapsin and the postsynaptic marker PSD95 on Apoer2 knockout mouse neurons rescued with lentiviral human APOER2-FL, control-specific APOER2 Δex4-5, +ex6B, Δex18 or AD-specific APOER2 Δex4-5, +ex6B, Δex15 isoform at 14 DIV where the only difference between these two APOER2 variants is either exclusion of ex15 or ex18. We measured the number of synapsin and PSD95 puncta independently and the number of synapses defined by the colocalization of synapsin and PSD95 (Figure 7).…”

“…Interactions between ligands and receptors such as APOER2 do not appear to be explained by simple binding patterns to single ligand-binding repeats, as exon-skipping removes individual domains but also changes overall structure and folding of the receptors. We observed that some exon skipping events in APOER2 lead to increased CTF formation, while others lead to decreased CTF formation, suggesting that receptor folding is a key factor in determining function (Omuro et al, 2022).…”

“…Next, we tested whether APOER2 cleavage by γ-secretase can be induced in a ligand-regulated manner by binding APOE. We have previously shown that APOE mimetic peptide (133-149 residues) derived from the receptor binding region, influences APOER2 splice variant receptor processing (Omuro et al, 2022). HEK293T cells were transfected with individual APOER2 isoforms for 24 hours and treated with 50 µM of APOE mimetic peptide for 30 min.…”

“…In humans, multiple alternative splicing events within APOER2 have been described in the brain (Clatworthy et al, 1999; Kim et al, 1997, 1996, Omuro et al, 2022), and unbiased RNA sequencing (RNAseq) studies on human brain tissue (Genotype-Tissue Expression [GTEx] Portal, NIH Common Fund) have expanded this diversity in annotated APOER2 splicing events. Using single molecule, long-read RNAseq, we recently identified a number of diverse and novel human APOER2 isoforms in the cerebral cortex that arise from a plethora of splicing combinations across the entire APOER2 transcript indicating possible differential functional effects at the protein level (Gallo et al, 2022).…”