Introduction: Apolipoprotein E4 (APOE4), the major genetic risk factor for Alzheimer’s Disease (AD), yet increasing evidence suggest APOE4 may detrimentally alter neurophysiology independent of classical AD hallmarks(1). Whilst hippocampal synaptic dysregulation caused by APOE4 is well-studied, the impact of APOE4 on intrinsic neuronal membrane properties is poorly studied.
Aims/Objectives: This study then looks to functionally characterise the impact of Apolipoprotein E genotype (APOE) on intrinsic neuronal excitability.
Methods: APOE-targeted replacement(2) mice were housed in 12/hr light/dark cycles with ad libitum water and food. Organotypic hippocampal slices were prepared as previously described(3) before patch-clamp electrophysiology was performed at DIV21. A current-step protocol from -200 pA to +400 pA allowed visualisation and analysis of intrinsic membrane properties(4). Modulation of the NMDAR was made separately with the following drugs (in µM): 20 ketamine, 10 memantine hydrocholride, 5 D-APV, 5 GNE-9278. Analysis of the relationship between APOE genotype and drug condition was inferred through multilevel linear modelling and bootstrap resampling (10,000 resamples). Post-hoc ANOVA permutation testing then analysed the interaction term. 12 animals per genotype were included, with a minimum of four slices per animal being produced. Only one recording per cell was then included in the analysis.
Results: Depolarizing current injection into CA1 neurons of APOE3 slice cultures caused neurons to fire at frequencies up to 11.00 Hz (95% CI [9.31, 12.31]). However, in APOE4 neurons, the firing rate was -2.94 Hz (95% CI [-5.06, -0.78], p = .007) lower. While acute application of ketamine had little effect in APOE3 neurons (+0.08 Hz, 95% CI [-2.94, +2.72], p = .95), ketamine increased firing rates more in APOE4 (than in APOE3) neurons by +3.53 Hz (95% CI [+0.15, +7.18], p = .043). Similarly to ketamine, acute application of memantine had little effect in APOE3 neurons (+0.71 Hz, 95% CI [-0.98, +2.65], p = .44) and increased firing rates more in APOE4 (than in APOE3) neurons by +4.27 Hz (95% CI [+1.00, +7.24], p = .021). A two-way ANOVA permutation test confirmed a significant interaction: the effect of the drugs (ketamine or memantine) on maximum firing rate depended on the APOE genotype (p = .023). To then confirm whether these described effects were mediated by the NMDAR a more specific NMDAR antagonist was applied, D-APV. While acute application of D-APV had little effect in APOE3 neurons (+1.88 Hz, 95% CI [-1.16, 5.29], p =.216), D-APV increased firing rates more in APOE4 (than in APOE3) neurons by +8.15 Hz (95% CI [ 3.35, 12.87], p = < .001). Further confirming the involvement of the NMDAR, acute application of the positive allosteric modulator, GNE-9278 significantly decreased firing rates in APOE3 neurons (-5.51 Hz, 95% CI [-8.06, -2.66], p = < .001) and decreased firing rates more in APOE3 (than in APOE4) neurons by -5.89 Hz (95% CI [ -1.16, -10.56], p = .016).
Conclusion: Not only does APOE4 perturb action potential firing, but the rescue of this phenotype is also dependent on the NMDAR hinting at a conserved pathway that could act as a future therapeutic target.