Activity-dependent neuroprotective protein (ADNP) exhibits striking sexual dichotomy impacting on autistic and Alzheimer's pathologies

Abstract

Activity-dependent neuroprotective protein (ADNP) is a most frequent autism spectrum disorder (ASD)-associated gene and the only protein significantly decreasing in the serum of Alzheimer's disease (AD) patients. Is ADNP associated with ASD being more prevalent in boys and AD more prevalent in women? Our results revealed sex-related learning/memory differences in mice, reflecting hippocampal expression changes in ADNP and ADNP-controlled AD/ASD risk genes. Hippocampal ADNP transcript content was doubled in male vs female mice, with females showing equal expression to ADNP haploinsufficient (ADNP(+/)(-)) males and no significant genotype-associated reduction. Increased male ADNP expression was replicated in human postmortem hippocampal samples. The hippocampal transcript for apolipoprotein E (the major risk gene for AD) was doubled in female mice compared with males, and further doubled in the ADNP(+/-) females, contrasting a decrease in ADNP(+/-) males. Previously, overexpression of the eukaryotic translation initiation factor 4E (eIF4E) led to ASD-like phenotype in mice. Here, we identified binding sites on ADNP for eIF4E and co-immunoprecipitation. Furthermore, hippocampal eIF4E expression was specifically increased in young ADNP(+/-) male mice. Behaviorally, ADNP(+/-) male mice exhibited deficiencies in object recognition and social memory compared with ADNP(+/+) mice, while ADNP(+/-) females were partially spared. Contrasting males, which preferred novel over familiar mice, ADNP(+/+) females showed no preference to novel mice and ADNP(+/-) females did not prefer mice over object. ADNP expression, positioned as a master regulator of key ASD and AD risk genes, introduces a novel concept of hippocampal gene-regulated sexual dimorphism and an ADNP(+/-) animal model for translational psychiatry.

Figure 1

ADNP^+/− differ from ADNP^+/+ male mice: object recognition, social interactions and sexual dichotomy. Animal performance in the object recognition test is shown (n=16–18 for each of the male groups; n=11 for each of the female groups). (a, b) ADNP^+/− male mice are deficient in object recognition. Two identical objects were first presented, and one of the identical objects was replaced by a novel object 3 h after sniffing the familiar object (short retention choice) (a) or on the following day (~24 h later, long retention choice) (b). Data are expressed as mean (±s.e.m.) by a relative discrimination index (D2=(b−a)/(b+a); b=time (s) sniffing a novel object, a=time (s) sniffing a familiar object). Two-way analysis of variance (ANOVA) revealed no significant differences in the short retention choice (a). In the long retention choice (b), two-way ANOVA showed a significant effect of genotype only in the male group (F(1,49)=5.022, P=0.030). ADNP-deficient male mice spent significantly less time (>2-fold) in exploring the new object as compared with control mice (ADNP^+/+). Fisher's LSD post hoc test revealed a significant difference between ADNP^+/− male mice compared with ADNP^+/+ mice (*P<0.05). (c) Sniffing time of empty cup and novel mouse—social recognition test. Data are expressed as mean (±s.e.m.) total time (s) spent exploring mice or objects. A three-chamber cage was used. Two-way repeated measure ANOVA with group as a fixed factor and sniffed item (that is, mouse vs cup) as repeated factor revealed no main effect for group (F(3,48)=1.051, P=0.379) on the sniffing time periods of a novel mouse and a cup. However, a main effect was found for the sniffed item (F(1,48)=75.761, P<0.001), indicating a strong preference for the novel mouse over the cup. In addition, an interaction effect between group × sniffed item was found (F(3,48)=3.296, P=0.028). Fisher's LSD post hoc test revealed significant differences between sniffing time period of the cup and mouse in all the groups (^*P<0.05, ^***P<0.001 vs cup in the same group—male or female). Bonferroni post hoc test revealed significant differences between sniffing time period of the cup and mouse in all the groups (^**P<0.01) except for ADNP^+/− females (P=0.18). The sniffing time of mouse in the latter group was significantly lower than in the ADNP^+/− males (^#P<0.05), with no change in the sniffing time of the cup (P>0.99 after adjustment for multiple comparisons). (d) ADNP^+/− mice displayed a significant decrease in social memory. Animal performance in the social memory test is shown (3 h after the original 3-min exposure). Data are expressed as mean (±s.e.m.) total time (s) spent exploring another mouse as designated by a relative discrimination index (b=time sniffing a novel mouse, a=time sniffing a familiar mouse. The total time allowed for sniffing in the second exposure was 3 min). In the social memory test, the ADNP-deficient male and female mice spent significantly less time exploring the novel mouse as compared with control mice (ADNP^+/+). Two-way ANOVA showed a significant genotype effect (F(1,47)=31.357, P<0.001) in the social memory test (ADNP^+/+ vs ADNP^+/− mice), but no general sex effect (males vs females), (F(1,47)=1.563, P=0.217). Fisher's LSD post hoc test revealed a significant genotype difference between the ADNP^+/+ and ADNP^+/− in the male group (^***P<0.001) as well as in the female group (^*P<0.05); there was also a significant sex effect in the ADNP^+/+ group (^*P=0.05 male vs female). ADNP, activity-dependent neuroprotective protein; LSD, least significant difference.

Figure 2

Odor habituation/dishabituation is intact in the ADNP^+/− ICR model exhibiting sexual dichotomy. Each mouse was tested during three consecutive 2-min periods for each odor with 2-min intervals between presentations. The time that the mouse smelled the swab was recorded (beginning when the animal oriented its nostrils toward the odor-scented cotton swab, within 2 cm or less). Male ADNP^+/+ mice performed better in the odor habituation/dishabituation test compared with female ADNP^+/+ mice. ^***P<0.001, ^*P<0.05 vs female at the same trial, t-test. ^##P<0.01 vs previous sniffing (novel vs familiar odor). ADNP, activity-dependent neuroprotective protein.

Figure 3

Identification and verification of ADNP and eIF4E interacting-sites. (a) Diagram of the predicted eIF4e protein binding motifs on human ADNP.^, (b) ADNP and eIF4E interaction. Co-immunoprecipitation assays were used to analyze ADNP and eIF4E binding. Lanes 1,5: 20 μg of brain lysate from ADNP^+/+ male mice and lanes 2,7: 20 μg of brain lysate from ADNP^+/− male mice immunoblotted with anti-eIF4E antibody; lane 3: 500 μg of brain lysate of ADNP^+/+ male mice and lane 4: 500 μg of brain lysate of ADNP^+/− male mice were immunoprecipitated with anti-ADNP antibody and immunoblotted with anti-eIF4E antibody; lane 6: 500 μg of brain lysate of ADNP^+/+ and lane 8: 500 μg of brain lysate of ADNP^+/− mice were immunoprecipitated without an antibody and immunoblotted with anti-eIF4E antibody. ADNP, activity-dependent neuroprotective protein.

Figure 4

Autism-specific gene modulation in the hippocampus of ADNP^+/− mice: sexual dichotomy. Hippocampal RNA from 5- to 6-month-old mice was analyzed by quantitative real-time PCR (n=6–8 mice per group). (a) eIF4E: Two-way analysis of variance (ANOVA) showed no significant effect of sex (P=0.692) and genotype (P=0.137). However, as there was a marginal genotype trend, one-way ANOVA was also performed showing a significant increase in eIF4E transcripts in the ADNP^+/− male mice compared with ADNP^+/+ male mice (^#P<0.05). (b) ADNP: two-way ANOVA showed a significant effect of sex (F(1,15)=8.760, P=0.010) and a significant effect of genotype (F(1,15)=14.660, P=0.002). Fisher's LSD post hoc test revealed a significant 2-fold decrease in ADNP^+/− male mice compared with control mice (^***P<0.001). In addition, Fisher's LSD post hoc test revealed a significant 2-fold increase in ADNP^+/+ male mice compared with ADNP^+/+ female mice (^*P<0.05). (c) Human ADNP: Hippocampal human ADNP used before was analyzed comparing males and females. As there was no difference at the ADNP transcript levels between normal and schizophrenia subjects, the cohorts were pooled to obtain 22 men and six women. Results showed a statistically significant increase in men (Student's t-test, ^*P<0.05). (d) ADNP2: two-way ANOVA showed no significant effect of sex (P=0.977) and no significant effect of genotype (P=0.498). (e) ApoE: two-way ANOVA showed a significant effect of sex (F(1,23)=128.734, P<0.001) and a significant effect of genotype (F(1,23)=13.430, P=0.001). Fisher's LSD post hoc test revealed a significant 2-fold increase in ADNP^+/− female mice compared with ADNP^+/+ female mice (^***P<0.001). In addition, Fisher's LSD post hoc test revealed a highly significant decrease in male mice compared with female mice for both ADNP^+/+ and ADNP^+/− (^***P<0.001). One-way ANOVA showed a significant decrease in ADNP^+/− male mice compared with ADNP^+/+ mice (^#P<0.05). (f) Nine-month-old male mice, eIF4E: no specific genotype effect, unlike (a) 5- to 6-month old. ADNP, activity-dependent neuroprotective protein; LSD, least significant difference.

Figure 5

Neuroligins show sex-dependent expression. Hippocampal RNA from 5-to 6-month-old mice was analyzed by quantitative real-time PCR (n=6–8 mice per group). The 4 NGLN genes were compared. (a) NGLN1: two-way analysis of variance (ANOVA) showed a significant effect of sex (F(1,26)=16.754, P<0.001), but no significant effect of genotype (P=0.561). Fisher's LSD post hoc test revealed a significant difference between male mice compared with female mice for both ADNP^+/+ and ADNP^+/− (^*P<0.05). (b) NGLN2: two-way ANOVA showed no significant effect of sex (P=0.777) and no significant effect of genotype (P=0.450). (c) NGLN3: two-way ANOVA showed a significant effect of sex (F(1,24)=7.800, P=0.010), but no significant effect of genotype (P=0.771). Fisher's LSD post hoc test revealed a significant difference between ADNP^+/− male mice compared with ADNP^+/− female mice (*P<0.05). (d) NGLN4: two-way ANOVA showed a significant effect of sex (F(1,25)=12.258, P=0.002), but no significant effect of genotype (P=0.482). Fisher's LSD post hoc test revealed a significant difference between ADNP^+/− male mice compared with ADNP^+/− female mice (*P<0.05). (e) Nine-month-old male mice, NGLN1: no specific genotype effect, like (a) 5- to 6-month old. (f) Nine-month-old male mice, NGLN2. (g) Nine-month-old male mice, NGLN3. (h) Nine-month-old male mice, NGLN4. ADNP, activity-dependent neuroprotective protein; LSD, least significant difference.