Apolipoprotein E4 in Alzheimer’s disease: an experimental study exploring its potential as a therapeutic target and its identification via a new genotyping-through-phenotyping approach

Introduction Alzheimer’s Disease (AD) is the most common form of dementia affecting around 30% of people over the age of 80 and the fourth leading cause of death in developed countries. Apolipoprotein E4 (ApoE4) has been indicated in literature as the most important susceptibility gene for late-onset Alzheimer's disease. ApoE has crucial roles in the CNS as well as in the periphery in all its isotypes, but ApoE4 specifically is involved in processes that contribute to the development of Alzheimer's disease: facilitating of amyloid beta aggregation, impairing its clearance, and fostering the formation of neurofibrillary tau tangles. Thus, therapeutic approaches have to focus on the selective neutralisation of ApoE4 without affecting the function of the other isoforms of Apolipoprotein E. To this end, human antibodies specifically recognising the E4 isotype may pose a powerful strategy to offer therapy for people at risk at an early preclinical stage. Moreover, the increased risk for AD in individuals expressing ApoE4 leads to the need to recognize those individuals with ApoE4 homozygous and heterozygous genotypes. Here I explain the approach employed by the research group I was part of for the search of anti-ApoE4-antibodies, and I propose a novel genotyping through phenotyping method,personally developed, under the supervision of Dr. Emmenegger. Methods Therapeutic target: interrogation of an unselected patient cohort twenty-thousand patients for the presence of autoantibodies against the E3,E4,and a lipidated form of the E4 isotypes of ApoE,aiming to identify patients harbouring anti-ApoE4-specific autoantibodies. Utilizing plasma samples provided by the Institute of Clinical Chemistry of UZH, a microELISA antibody profiling was carried out in a robotised platform. Medical and experimental data was separately stored in an multi-database system and was correlated and visualised using software packages in Python and R. Phenotyping: ApoE4 isotypes in patients of interest was assessed using an in-house developed ApoE phenotyping approach. Results: Therapeutic target: Testing of minimally 13,741 samples per isotype indicates a prevalence of positives of 0.08% for E3, 0.01% for E4, and 0.04% for the lipidated form of E4. Then, those patients reactive against at least one of the isotypes were selected and tested against all others. Preliminary data from these secondary screenings suggest that few patients that selectively target either only the lipidated form of ApoE4 or bind E4 (lipidated and non-lipidated) with higher affinity than E3. The correlation of the medical record with the reactivity spectrum of the patients has not yielded a clear-cut pattern, possibly indicating that the presence of antibodies against ApoE is not associated with a specific disease type. Phenotyping: the phenotyping approach developed seems to be valid in differentiating samples that contain from those that do not contain ApoE4, thereby indicating those individuals who have at least one of two ApoE4 alleles. The efficacy in differentiating homozygous from heterozygous individuals is variable across assays. Conclusion Therapeutic target: While preliminary data indicate the possibility to identify patients harbouring ApoE4-specific antibodies, more sophisticated secondary screenings are needed to validate the findings. Next steps include, the attempt to clone antibodies from the memory B cells of those patients with ApoE4-specific antibodies, and the characterization of the antibodies in in vitro and in vivo disease models. The successful realisation of these steps may open up new avenues to help prevent the development of AD in susceptible patient groups at an early pre-symptomatic stage. Phenotyping approach: protocol for identification of ApoE4 is validated and stable, may need further optimization in order to differentiate homozygous from heterozygous individuals.