ApoE4 decreases spine density and dendritic complexity in cortical neurons in vivo

Abstract

The three human alleles of apolipoprotein E (APOE) differentially influence outcome after CNS injury and affect one's risk of developing Alzheimer's disease (AD). It remains unclear how ApoE isoforms contribute to various AD-related pathological changes (e.g., amyloid plaques and synaptic and neuron loss). Here, we systematically examined whether apoE isoforms (E2, E3, E4) exhibit differential effects on dendritic spine density and morphology in APOE targeted replacement (TR) mice, which lack AD pathological changes. Using Golgi staining, we found age-dependent effects of APOE4 on spine density in the cortex. The APOE4 TR mice had significantly reduced spine density at three independent time points (4 weeks, 3 months, and 1 year, 27.7% +/- 7.4%, 24.4% +/- 8.6%, and 55.6% +/- 10.5%, respectively) compared with APOE3 TR mice and APOE2 TR mice. Additionally, in APOE4 TR mice, shorter spines were evident compared with other APOE TR mice at 1 year. APOE2 TR mice exhibited longer spines as well as significantly increased apical dendritic arborization in the cortex compared with APOE4 and APOE3 TR mice at 4 weeks. However, there were no differences in spine density across APOE genotypes in hippocampus. These findings demonstrate that apoE isoforms differentially affect dendritic complexity and spine formation, suggesting a role for APOE genotypes not only in acute and chronic brain injuries including AD, but also in normal brain functions.

Figure 1.

APOE4 decreases spine density in pyramidal cells in cortical layers II/III at 4 weeks of age. Mouse brains were Golgi stained and cortical layers II/III were imaged (n = 4 mice/genotype). A, Representative Golgi-impregnated neuron. B, Representative AO and BS dendrites per genotype. C–E, Averaged spine densities for each genotype. C, Averaged total spine density, combining AO + BS dendrites (56 dendritic segments/group). D, Averaged spine density for AO spines (28 neurons/group). E, Averaged spine density for BS spines (28 neurons/group). F–H, The cumulative distribution percentage of spine length. APOE2 TR mice had a significant shift in distribution to longer spines compared with other groups (p < 0.05, Kolmogorov–Smirnov test). *P < 0.05.

Figure 2.

APOE isoforms exhibit similar spine density in the hippocampus at 4 weeks of age. Mouse brains were Golgi stained and granule cells in the dentate gyrus and CA1 neurons were imaged (n = 4 mice/genotype). A, Left, A Golgi impregnation of the hippocampus of an APOE3 TR mouse at 2.5×. Right, A representative image of APOE3 TR mouse granule cells in the dentate gyrus at 20× magnification. B, Representative dendrites for the dentate gyrus per genotype. C, Averaged total spine density in the dentate gyrus (28 neurons/genotype). D, The cumulative percentage distribution plots of spine length. E, Representative image of APOE3 TR CA1 neurons at 10× magnification. F, Averaged data include quantification of AO (28 neurons/genotype). G, BS (28 neurons/genotype) spine number per 10 μm length.

Figure 3.

ApoE4 further reduces spine density in the cortex at 1 year. A, Averaged total spine density (56 neurons/group). B, Averaged AO spine density (28 neurons/group). C, Averaged BS spine density (28 neurons/group). *p < 0.05 compared with APOE3. D, Cumulative distribution of spine length. E–G, Mouse brains were Golgi stained and cortical layers II/III imaged at 1 year of age (n = 4 mice/genotype). E, Averaged data include total spine density (56 neurons/genotype). F, Averaged data include AO spine density (28 neurons/genotype). G, Averaged data include BS spine density (28 neurons/genotype). *p < 0.05 compared with APOE3. H, Cumulative percentage distribution of spine length. APOE4 TR mice had a significant shift in distribution toward smaller spines compared with the other APOE TR (E3, E2) mice at 1 year of age. I, J, Mouse brains were Golgi stained and granule cells in the dentate gyrus were imaged at 3 months (n = 4). I, Averaged total spine density for all genotypes (28 neurons/group). J, Cumulative percentage distribution of spine length. K, L, Mouse brains were Golgi stained and granule cells in the dentate gyrus were imaged at 1 year (n = 4). K, Averaged total spine density (28 neurons/group). L, Cumulative percentage distribution for spine length. APOE2 TR had a significant shift in distribution toward shorter spines compared with APOE (E3, E4) TR mice.

Figure 4.

ApoE4 decreases dendritic arborization in cortical neurons at 4 weeks. A, Three-dimensional graphical tracing representing dendrite morphology. The number of apical and basal dendrites was measured in micrometers, and the number of apical and basal nodes was counted. B, Averaged data include the node (branch) number for apical and basal dendrites for the three genotypes, analyzing 18 neurons per group. APOE2 mice had increased branching compared with APOE3 (25.5 ± 14.5%) and APOE4 (39.8 ± 11.7%). C, Averaged total dendritic length for apical and basal dendrites for the three genotypes, analyzing 18 neurons per group. APOE2 mice had significantly increased length (29.6 ± 9.6) compared with APOE4 TR mice. *p < 0.05, **p < 0.01. D, Using Scholl analysis, we graphed the average intersections per shell per neuron against the distance from the soma (in micrometers). ^ap < 0.05 for APOE3 versus APOE2. ^bp < 0.05 for APOE4 versus APOE2.