Sexual dimorphism of AMBRA1-related autistic features in human and mouse

Abstract

Ambra1 is linked to autophagy and neurodevelopment. Heterozygous Ambra1 deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of AMBRA1 for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal AMBRA1 genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower AMBRA1 mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by in silico analysis. Searching for further autism-relevant characteristics in Ambra1^+/- mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an in vivo readout of neuronal excitation-inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of AMBRA1/Ambra1 partial loss-of-function genotypes for female autistic traits. Moreover, they suggest Ambra1 heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.

Figure 1

Human AMBRA1-rs3802890 G/A: association with autistic features. (a) Quantification of autistic symptoms using PAUSS (PANSS Autism Severity Score. Note the high intercorrelation of PAUSS items and the high internal consistency of the scale (Spearman rank correlation coefficients; Cronbach’s α). (b) PGAS using AMBRA1-rs3802890 and PAUSS score: female AA subjects display a higher PAUSS score than GG subjects in the discovery sample; mean±s.e.m.; two-tailed Mann–Whitney U-test (data-corrected by linear regression analysis for age). (c) Trends of positive association between rs3802890-AA genotype and sub-items of PAUSS, more pronounced in females; mean±s.e.m.; two-tailed Mann–Whitney U-test. (d) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype; mean±s.e.m. (e) Genotype effect of AMBRA1-rs3802890 on degree of social support in the discovery sample, again significant in females; mean±s.e.m.; Mann–Whitney U-test. (f) Replication of the genotype and gender effect of AMBRA1-rs3802890 using social support as proxy in the general population; linear regression analyses (bootstrap; data-corrected for age); mean±s.e.m. (g) Relative AMBRA1 mRNA expression in peripheral blood mononuclear cells (PBMC) is reduced in female AA (risk SNP) carriers: shown is the AMBRA1 mRNA expression in AA carriers, given as mean Δ-value compared to GG carriers (GG males N=33; GG females, N=14). Values of individual AA males (N=35) and AA females (N=33) are expressed in %GG (mean) of the respective gender. The Δ-value is calculated as: Δ=%GG–100% mean±s.e.m.; two-tailed Mann–Whitney U-test.

Figure 2

AMBRA1-rs3802890 G/A: in silico approach to mechanistic insight. (a) Detailed map showing the human AMBRA1 gene (NCBI-accession: 55626; chromosomal location: Chr.11: 46 396 412–46 594 069). The AMBRA1 mRNA (21 exons) is mapped at the bottom. The location of AMBRA1-rs3802890 is indicated by a red asterisk. The region shared between all chromosomes (nucleotides from around 9530 to 11 030 on AMBRA1; red arrow) is indicated. Similar to man, the murine Ambra1 gene region matches on all chromosomes (nucleotides from around 68 110–69 940 on murine Ambra1). However, the chromosomal match region of AMBRA1/Ambra1 is different between both species. Both match regions show similarities to expressed sequence tags (EST) from NCBI database (best match EST gi|22688027 in human and gi|44663783 in mice; both with full-length alignment) and to specific lncRNAs in Refseq database (best match human: Refseq Accession NR_126435.1 named LINC00504; best match in mice: Refseq Accession NR_131899.1 named Mrqpra6). (b) RNA folding of human AMBRA1 transcribed EST comparing the G allele with the A allele. The presentation of thermodynamic ensemble folding stresses differences in the obtained structure. The color code indicates pairing propensities. The fold is further supported by reoccurring similar differences comparing several foldings and also lengths using software mFold. As template for folding, the full RNA sequence of EST from myelogenous leukemia cells (496 nucleotides long, 98% identity; full-length alignment; genbank accession BM149074.1; pediatric acute myelogenous leukemia cell (FAB M1) EST TCAAP2E6309) is shown. This RNA encodes no protein, has no introns/exons and has no complementary match in any other chromosomal region. (c) EST TCAAP2E6309 expression in all tested tissues: RNA was isolated from peripheral blood mononuclear cells (PBMC), human glioblastoma tissue (GB), and human placenta (PL). PCR was performed from cDNA. To exclude false positive results by genomic DNA contamination, several controls were performed (DNase digestion; respective RNA amplification). Genomic DNA from whole blood was used as positive control (+) and ddH₂O as negative control (−).

Figure 3

Impaired pheromone preference in female Ambra1 mutants. (a) Intellicage apparatus with connected social boxes. (b) Time spent in social boxes with used or fresh bedding or delta difference scores for the indicated genotypes. Upper row females; lower row males; mean±s.e.m. presented. Within-group comparisons performed with paired t-tests, between-group with Mann–Whitney U-tests; all tests two-tailed.

Figure 4

Brain enlargement in prepubertal Ambra1 mutants of both genders. Shown are results of high-resolution magnetic resonance imaging (MRI) (T2-weighted). Brain regions of interest (whole brain, hippocampus, cerebellum, olfactory bulb, ventricles) in 23day-old female (upper row) and male (lower row) mice of both genotypes are presented; mean±s.e.m.; two-tailed unpaired t-tests. Right side: Representative pictures of 3D-reconstructed brains of both genotypes illustrate brain enlargement in mutants.

Figure 5

Persistent brain enlargement and altered pentylentetrazol (PTZ)-induced seizure propensity in female Ambra1 mutants. (a) Brain regions of interest (whole brain, hippocampus, cerebellum, olfactory bulb, ventricles) were analyzed by high-resolution magnetic resonance imaging (MRI) in 13-months-old female Ambra1^+/− and WT mice; mean±s.e.m.; two-tailed unpaired t-tests. (b) PTZ-induced seizure propensity (intraperitoneal injection of 50 mg kg⁻¹) in 23-day-old WT and Ambra1^+/− mice of both genders; mean±s.e.m. presented; two-tailed unpaired t-tests. (c) PTZ-induced seizure propensity (intraperitoneal injection of 50 mg kg⁻¹) in 13-months-old female WT and Ambra1^+/− mice; mean±s.e.m. presented; two-tailed unpaired t-tests. (d) Survival of PTZ-induced seizures (intraperitoneal injection of 50 mg kg⁻¹) in 13-months-old female WT and Ambra1^+/− mice; Kaplan–Meier survival analysis.