In a recent study Nature is neuroscienceResearchers investigated whether the R136S genetic mutation protected against apolipoprotein E4 (APOE4)-induced late-onset Alzheimer’s disease (LOAD).
Study: APOE-R136S mutation protects against APOE4-driven tau pathology, neurodegeneration and neuroinflammation. Image credit: Atthapon Rakshaput/Shutterstock.com
APOE4 may increase the risk of LOAD, which may exacerbate earlier clinical onsets and pathologies, including amyloid-beta and tau protein deposition, neuroinflammation, and neurodegeneration.
APOE4 carriers contribute to 55% to 75% of Alzheimer’s disease cases, emphasizing the importance of investigating their role in Alzheimer’s disease pathogenesis and identifying protective targets. The rare APOE3-R136S variant was found to protect against early-onset AD in PSEN1-E280A carriers.
About the study
In the current study, researchers investigated the potential of the APOE-R136S mutation to protect against apolipoprotein E4-induced LOAD.
The research team conducted an experiment in which they inserted R136S into the apolipoprotein E4 allele of human apolipoprotein E4 knock-in (E4-KI) mice and human-induced pluripotent stem cells (hiPSCs) obtained from an AD patient.
The team next studied the effects of this document in hiPSC-derived neurons to investigate its potential to protect against APOE4-induced tau pathology. Five isogenic hiPSC lines were karyotyped and differentiated into neurons expressing microtubule-associated protein 2 (MAP2).
The study used previously generated isogenic APOE3 hiPSC lines (E3), parental E4 hiPSC lines (E4), and isogenic hiPSC-derived neurons, including isogenic homozygous (E4-S/S) and heterozygous (E4-R/S). line line
The researchers introduced R136S into the human E4-knockout APOE4 locus in murine animals using CRISPR-Cas-9-mediated gene editing. Immunocytochemistry was used to monitor hippocampal phosphorylated tau (p-tau) protein levels. At the same time, the effects of R136S on human neuronal cells were evaluated by quantifying APOE levels by western blotting.
Additionally, the effects of heterozygous and homozygous R136S mutations on tau uptake were assessed using human neuronal cells. Flow cytometry determined differences in Tau-488 protein internalization by human neuronal cells with different APOE genotypes.
The team measured the number of Tau-488-expressing cells to investigate whether reduced uptake of tau by heparin sulfate proteoglycan (HSPG) contributed to the prevention of APOE4-induced accumulation of p-tau protein. They also investigated whether R136S protected against APOE4-promoted neurodegeneration in PS19-E mice.
The researchers surveyed the hippocampus to measure astrocyte coverage as a percentage and normalized the astrocyte count to the size of the hippocampal region.
They examined the level of hippocampal microgliosis in PS19 mice with different APOE genotypes using ionized calcium-binding adapter molecule 1 (Iba1) and cluster of differentiation 68 (CD68) staining. The effect of R136S on the transcriptomic signature of the hippocampus was examined at a cellular level.
Tauopathy mice were formed expressing human APOE4 or APOE4-R136S. R136S protected against APOE4-induced p-tau protein build-up and progressive p-tau protein staining in a murine tauopathy model.
The homozygous R136S mutation reduced tau pathology in tauopathy mice and only the homozygous R136S mutation protected against APOE4-induced p-tau protein build-up in human neurons. The homozygous R136S mutation reduces HSPG-mediated tau uptake by human neurons, likely due to defective HSPG binding and lack of APOE in the homozygous mutation, respectively.
The presence of homozygous and heterozygous R136S mutations protects against APOE4-induced astrocytosis and microgliosis, known to cause hippocampal neurodegeneration and atrophy in the context of tauopathies. This protection was age-dependent, with homozygous mutations being more effective than heterozygous mutations.
The homozygous R136S mutation also effectively reduces the build-up of p-Tau protein induced by APOE4 in the hippocampus. In contrast, the heterozygous mutation only partially protects against apolipoprotein E4-induced neurodegeneration and neuroinflammation but not tau pathology.
Furthermore, the homozygous mutation model showed a fivefold increase in APOE protein levels in E4 neurons, indicating that R136S may help reduce the deleterious effects of apolipoprotein E4 on gamma-aminobutyric acid (GABA)-ergic neurons.
APOE expression by MAP2-positive neurons was verified by immunocytochemistry, and neuronal cultures were verified negative for non-neuronal cells including oligodendrocytes, neural stem cells and astrocytes, oligodendrocyte transcription factor (Olig2), sex-determining region Y-determining 2 respectively. (SOX2), and glial fibrillary acidic protein (GFAP) levels.
E3 neurons have significantly reduced p-tau levels compared to E4 neurons. Neurons with the homozygous mutation showed a 50% decrease in mean fluorescent intensity (MFI) compared to E4 neurons, indicating reduced Tau-488 protein uptake.
Echo-type neurons significantly decreased Tau-488 protein uptake vs . E4 neurons, indicating that R136S has reduced apolipoprotein activity that functionally contributes to Tau-488 accumulation in neuronal cells with mutations homologous to echo-type neurons.
Heparin therapy significantly reduced mean fluorescent intensity in neuronal cells with E3, E4 and heterozygous mutations. In contrast, heparin levels only tended to decrease in homozygous E4 mutated-neuronal cell MFI, indicating reduced HSPG-based tau protein uptake in neuronal cells with homozygous mutations.
Based on the results of the study, the presence of the R136S mutation in the APOE4 gene was found to protect the mice against the production of tau protein and the development of tauopathy.
This mutation exhibits a dose-dependent effect, potentially a protective factor against Alzheimer’s disease.
- Nelson, M.R., Liu, P., Aggarwal, A., etc(2023) APOE-R136S mutation protects against APOE4-driven tau pathology, neurodegeneration and neuroinflammation, Nat Neurosci. doi: https://doi.org/10.1038/s41593-023-01480-8. https://www.nature.com/articles/s41593-023-01480-8