Cognitive deficits and disruption of neurogenesis in a mouse model of apolipoprotein E4 domain interaction

Abstract

Apolipoprotein E4 (apoE4) allele is the major genetic risk factor for sporadic Alzheimer disease (AD) due to the higher prevalence and earlier onset of AD in apoE4 carriers. Accumulating data suggest that the interaction between the N- and the C-terminal domains in the protein may be the main pathologic feature of apoE4. To test this hypothesis, we used Arg-61 mice, a model of apoE4 domain interaction, by introducing the domain interaction feature of human apoE4 into native mouse apoE. We carried out hippocampus-dependent learning and memory tests and related cellular and molecular assays on 12- and 3-month-old Arg-61 and age-matched background C57BL/6J mice. Learning and memory task performance were impaired in Arg-61 mice at both old and young ages compared with C57BL/6J mice. Surprisingly, young Arg-61 mice had more mitotic doublecortin-positive cells in the subgranular zone; mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB were also higher in 3-month-old Arg-61 hippocampus compared with C57BL/6J mice. These early-age neurotrophic and neurogenic (proliferative) effects in the Arg-61 mouse may be an inadequate compensatory but eventually detrimental attempt by the system to "repair" itself. This is supported by the higher cleaved caspase-3 levels in the young animals that not only persisted, but increased in old age, and the lower levels of doublecortin at old age in the hippocampus of Arg-61 mice. These results are consistent with human apoE4-dependent cognitive and neuro-pathologic changes, supporting the principal role of domain interaction in the pathologic effect of apoE4. Domain interaction is, therefore, a viable therapeutic/prophylactic target for cognitive impairment and AD in apoE4 subjects.

Keywords: Alzheimer Disease; Antagonistic Pleiotropy; ApoE; Apoptosis; BDNF; Doublecortin; Learning and Memory; Neurogenesis; Protein Domains.

FIGURE 1.

Impaired spatial recognition memory and cognitive flexibility in 12-month-old Arg-61 mice. a, radial-arm water maze tank with dimensions. Shown are average time-to-target (b) and errors made during the first (white dots with black background) and fourth (dotted squares with white background) block of trials on day 2 (c). Both C57BL/6J and Arg-61 mice seemed to have learned the location of the platform as there were reductions in time to target and errors made during the first block of trial versus the fourth (paired sample t test). There was no significant difference between C57BL/6J and Arg-61 mice in both measures during both of the trial blocks. Shown are time to locate the target (d), total errors or wrong arm entries made (e), and number of entries made into the previous target arm during the reversal learning (a test of cognitive flexibility) when the target with which they were trained for 2 days was changed to another one (f). Shown are percent time (g) and percent of total entries (h) in the Novel arm during a place memory/novel arm discrimination test. During the retention trial, mice were allowed to explore the three arms of a Y-maze that they had explored 4 h earlier during a 10-min acquisition trial when one of the arms (The Novel arm) was blocked. C57BL/6J mice discriminated between the novel and familiar arms, as the percent time and entry in the novel arm were significantly higher than the 50% chance level (dotted) line. Although the percent duration in the novel arm was not significantly different (p > 0.05), the percent entry into the novel arm was significantly higher in C57BL/6J mice than the Arg-61 mice (p = 0.0498; §). For a and b, n = 7 for both groups. Data shown are the mean ± S.E. One sample t test (versus 50%). *, p < 0.05; **, p < 0.01. For c–e, n for C57BL/6J was 18; n for Arg-61 was 14. Independent sample t test (C57BL/6J versus Arg-61). *, p < 0.05; **, p < 0.01.

FIGURE 2.

Impaired spatial learning and memory in 3-month-old Arg-61 mice. Shown are latency/time-to-target (a) and total number of errors/wrong arm entries (b) made on day 1 of the radial-arm water maze test. No significant difference in number of errors but latency was significantly higher in Arg-61. Analysis of the total distance swum and swimming speed in the subsequent probe trial shows no significant difference in both locomotor measures (data not shown; see “Results”). Independent sample t test (C57BL/6J versus Arg-61). *, p < 0.05; **, p < 0.01. n for both groups was 10 (5 males, 5 females). Training blocks were two consecutive trials with two different start arms for each mouse. Shown are percent of time (c) and percent of entry in the target arm (d) during a series of probe trials in the radial arm water maze when the target was removed. Data shown are the mean ± S.E. One-sample t test versus 20% chance (dotted line) level. *, p < 0.05; **, p < 0.01. n for both groups is 10 (5 males and 5 females). No significant difference between C57BL/6J and Arg-61 mice (independent t test p > 0.05) was found on any of the days in either percent duration (c) or frequency (d) in target. However, there was a trend to a higher percent time in target c in C57BL/6J mice on day 5 (p = 0.0693). Day 1 = 1 h after the last trial on day 1; day 2 = 1 h after the last trial on day 2; day 3 = 24 h after the last trial on day 2; day 5 = 72 h after last trial on day 2.

FIGURE 3.

Impairment in young mice is gender-dependent; females more affected. a, average time-to-target over the five training blocks among 3-month-old male and female C57BL6J and Arg-61 mice. There was no significant difference among males, but average time to target was significantly different among females p = 0.025. b, spontaneous alternation percentage score for 3-month-old male and female mice. All groups performed beyond chance level (50%; dotted line) except the female Arg-61 mice. Two-way analysis of variance shows a significant gender effect (F(1,16) = 6.070; §, p = 0.0255); although both t test and post hoc test did not reveal a significant male versus female difference in either genotype (p > 0.05), the t test showed a trend to a significantly lower alternation percentage in female Arg-61 mice versus males (p = 0.0591) but not in C57BL/6J mice (p = 0.226). Shown is percent of time (c) and percent of total entries (d) in the Novel arm during a place memory/Novel arm discrimination test. During the retention trial mice were allowed to explore the three arms of a Y-maze, which they had explored 1 h earlier during a 5-min acquisition trial when one of the arms (novel arm) was blocked. Female C57BL/6J mice discriminated between the novel and familiar arms, as the percent time and entry in novel arm were significantly higher than 50% chance level (dotted) line, whereas female Arg-61 mice did not; the percent time in novel arm was not significantly different between C57BL/6J and Arg-61 (p > 0.05), but there was a trend to a higher percent entry into novel arm in C57BL/6J mice versus Arg-61 mice (p = 0.0524). Data shown are the mean ± S.E. One-sample t test versus 50% chance (dotted line) level. *, p < 0.05; **, p < 0.01. n for all groups is 5.

FIGURE 4.

Increased doublecortin-positive cells in the dentate gyrus of 3-month-old Arg-61 mice. Doublecortin-positive cells were found in the neurogenic regions of the brain, namely the sub-ventricular zone along the walls of the lateral ventricles and the subgranular zone of the hippocampus where they were found lining the junction of the hilus and granular layer. Some cells were found within the granular cell layer where they are located mostly closer to the hilus than the molecular layer. a, mitotic and post-mitotic doublecortin positive cell numbers in the dentate gyrus; cells were classified based on the descriptions by Plümpe et al. (31); see “Results.” b, total doublecortin-positive cells in the dentate gyrus of C57BL/6J and Arg-61 mice. Data shown are the mean ± S.E.; n = 4 for both groups. Representative sample images of doublecortin-positive cells in the dentate gyrus of C57BL/6J (c) and Arg-61 mice (d) are shown. Images taken with a ×10 objective; scale bar, 100 μm. Samples of doublecortin-positive cells in different classifications, namely postmitotic (e) and intermediate (f) and mitotic (g) types (also indicated by asterisks in f. Images were taken with a ×40 objective; scale bar, 10 μm.

FIGURE 5.

Potential/possible neurotropic and apoptotic processes in Arg-61 mice. Shown is hippocampal expression of BDNF (a and c) and TrkB (b and d) mRNA in the hippocampus of 3- and 12-month-old C57BL/6J and Arg-61 mice, respectively. Although young Arg-61 mice had significantly higher mRNA levels of both BDNF (a) and TrkB (b) in their hippocampi (*, p = 0.0318 and 0.0291 respectively; n = 4 for C57BL/6J and 5 for Arg-61), there were no significant differences in the mRNA levels of both genes between 12-month old C57BL/6J and Arg-61 mice (p > 0.05 for both BDNF and TrkB; n = 5 for both C57BL/6J and Arg-61). Despite the higher mRNA levels of BDNF in young Arg-61 mice, protein levels of BDNF in both young (e) and old mice (f) showed no significant differences between C57BL/6J and Arg-61 mice (p > 0.05 in both cases. n = 4 for both genotypes of the young animals and 5 for both genotypes of the old animals). However, Arg-61 mice had higher levels of cleaved caspase-3 at both ages with an 87.7% increase (**, p = 0.006; n = 5 per group) in the young (g) and a much higher 172.4% increase (*, p = 0.0145; n = 5 per group) in the old (h) animals. All data are presented as the mean ± S.E. In all figures white bars represent C57BL/6J and black bars represent Arg-61 mice.

FIGURE 6.

Decreased doublecortin levels later in life in old Arg-61 mice. Doublecortin expression in the hippocampus of 3- and 12-month-old male C57BL/6J and Arg-61 mice. Data represent the mean ± S.E.; *, p = 0.002; n = 3 in all groups except 12-month-old Arg-61 in which n = 4.