Studying the Effect of Apoe mRNA Isoforms on Alternative Splicing and Alzheimer’s Disease Pathology

Alzheimer’s disease (AD) is the primary form of dementia, accounting for between 60-80% of all cases, and its prevalence is increasing dramatically worldwide due to the combination a rapidly aging population combined with a lack of disease-modifying therapeutics. Notably, despite significant efforts, the cause of Alzheimer's disease is still poorly understood. However, it is widely agreed upon that aging and possession of the APOE epsilon 4 (apoE4) allele are the major purported risk factors.

APOE is a polymorphic gene in humans encoding the lipid and cholesterol transporter apolipoprotein E (apoE). Relative to the most common isoform, apoE3, it is well characterized that the apoE4 isoform exacerbates the risk for cardiovascular disease, late-onset Alzheimer’s disease (LOAD), and cognitive decline during aging. However, precise mechanisms by which apoE isoforms differentially contribute to brain health during aging remain unclear.

In this paper, we sought to characterize the effect of apoE mRNA isoforms on alternative splicing and Alzheimer’s disease pathology by elucidating the correlation of apoE mRNA isoforms with previously characterized neurodegenerative phenotypes and identifying distinct upstream regulators of apoE mRNA splicing in healthy and diseased tissue.

Experiments in primary dissociated cultures of apoE genetic knockout astrocytes transduced with lentiviral vectors, each expressing the ApoE open reading frame of a single mRNA isoform, were used in conjunction with pooled genetic screens with scRNA-seq readout to identify the function of each apoE mRNA isoform in Alzheimer’s disease processes. Additionally, we conducted a combination of ir-CLIP and ChIRP-MS to characterize any discrepancies in pre-mRNA splicing between healthy and diseased tissue. Through these experiments and subsequent downstream analyses, we identified two mRNA isoforms that were differentially up regulated in Alzheimer’s disease processes and four potential splicing factors with associated pre-mRNA sequences implicated in splicing regulation for diseased brain tissue.

This work uncovers a better understanding of how various APOE mRNA transcriptional isoforms impact brain health and which splicing factors are involved in this gene-splicing regulation to hopefully inform the development of future novel neuroprotective agents and targets.