Identification of novel apolipoprotein E receptor 2 splice variants and their role in synaptic transmission

Apolipoprotein E (APOE) is one of the most important genetic risk factors for late-onset sporadic Alzheimer’s disease (LOAD). APOE is a 35 kDa glycoprotein and ligand known to bind to members of the low-density lipoprotein receptor (LDLR) family, including APOE receptor 2 (apoER2; official gene name LRP8). ApoER2 is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail that can be proteolytic cleaved. In addition, apoER2 is enriched in the brain and plays an important role in synaptic function and plasticity. Interestingly, the ECD of apoER2 contains several ligand binding repeats that are organized into exons with aligning phase junctions, which allows exon skipping during alternative splicing to retain protein fidelity. The amount of alternative spliced isoforms distinguishes apoER2 from the rest of the LDLR family members. In fact, mouse Apoer2 has been identified as one of the top ten neuronal genes related to cell-type exon skipping events. Regarding human APOER2, we have identified over 40 different APOER2 isoforms from human brain using gene-specific primers and amplifying the N- and C-terminal open reading frame of APOER2. The majority of APOER2 variants consist of a diverse array of exon skipping events within the ligand binding domain (LBD). We therefore, hypothesized that human APOER2 splice variants act as functionally divergent isoforms that can influence ligand binding properties, receptor proteolysis and changes to synaptic function. ApoER2 undergoes sequential proteolytic cleavage in response to ligand binding, resulting in the release of C-terminal fragments (CTFs) and transcriptionally active intracellular domain (ICD). We therefore, systematically tested whether the diversity of human N-terminal APOER2 splice variants lacking various LBDs affects APOER2 cleavage and signaling events. We found that alternative splicing of certain APOER2 exons generated different amounts of CTFs compared to full-length APOER2 (APOER2-FL). The pattern was not simply based on the number of ligand binding domains suggesting that excision of certain exons may alter the tertiary structure of the receptor sufficiently to make the receptor more or less accessible to cleavage and generation of CTFs. To further characterize APOER2 splice variants, we specifically examined APOER2 splice variants that generated the highest and lowest amounts of CTF generation compared to APOER2-FL and focused on APOER2 splice variant lacking exons 5-8 (Δ5-8) and lacking exons 4-6 (Δ4-6), respectively. The differential CTF generation of APOER2 Δ5-8 and Δ4-6 reflects the proteolytic release of the APOER2-ICD. This APOER2-ICD mediates transcriptional activation, facilitated by the Mint1 adaptor protein. To investigate whether human N-terminal APOER2 splice variants influence APOE binding and receptor cleavage properties, we used microscale thermophoresis and tested the well-validated human APOE mimetic peptide. We found that specific exons or ligand-binding cassettes differentially affect APOE peptide binding to APOER2 splice variants. In addition, APOE peptide induces generation of APOER2-CTF acutely within one hour. Functionally, we demonstrated that APOER2 is required for spontaneous neurotransmitter release in mature neurons. Loss of mouse Apoer2 robustly decreased miniature event frequency in excitatory synapses compared to heterozygous Apoer2 neurons. We found APOER2-FL fully restored the miniature event frequency in excitatory synapses but not APOER2 Δ5-8. APOER2 Δ4-6 restored the miniature event frequency similar to heterozygous Apoer2 neurons. These results suggest that different human N-terminal APOER2 splice variants have distinct and differential synaptic properties signifying a role of APOER2 splice variants as regulators of synaptic function