D-aspartate oxidase gene duplication induces social recognition memory deficit in mice and intellectual disabilities in humans

Abstract

The D-aspartate oxidase (DDO) gene encodes the enzyme responsible for the catabolism of D-aspartate, an atypical amino acid enriched in the mammalian brain and acting as an endogenous NMDA receptor agonist. Considering the key role of NMDA receptors in neurodevelopmental disorders, recent findings suggest a link between D-aspartate dysmetabolism and schizophrenia. To clarify the role of D-aspartate on brain development and functioning, we used a mouse model with constitutive Ddo overexpression and D-aspartate depletion. In these mice, we found reduced number of BrdU-positive dorsal pallium neurons during corticogenesis, and decreased cortical and striatal gray matter volume at adulthood. Brain abnormalities were associated with social recognition memory deficit at juvenile phase, suggesting that early D-aspartate occurrence influences neurodevelopmental related phenotypes. We corroborated this hypothesis by reporting the first clinical case of a young patient with severe intellectual disability, thought disorders and autism spectrum disorder symptomatology, harboring a duplication of a chromosome 6 region, including the entire DDO gene.

Fig. 1. Reduced gray matter volume in motor cortex and striatal regions of adult Ddo knockin mice and lower number of cortical neurons in the dorsal pallium of newborn pups.

a, b Representative images of sagittal sections showing Ddo expression in (a) R26^+/+ and (b) R26^Ddo/+ pups at birth. C cortex, E eye. Scale bar 1 mm. c High-resolution structural MRI revealed a prominent reduction of gray matter volume (GMV) in cortical and striatal areas of R26^Ddo/+ compared to R26^+/+ mice. d Regional volumetric analysis of GMV in R26^+/+ and R26^Ddo/+ mice in dorsal striatum (dStr), primary motor cortex (M1) and somatosensory cortex (SS). e–i Birth-dating experiment of embryos injected with BrdU at E14.5. e–h Representative coronal sections showing BrdU-positive neurons in (e, f) R26^+/+ and (g, h) R26^Ddo/+ mice at birth. f, h Confocal higher magnification of the boxed region in the dorsal pallium. Images further demonstrate the reduced density of BrdU-positive cells in (h) R26^Ddo/+ pups as compared to (f) R26^+/+ controls. Scale bars: (e, g) 250 μm, (f, h) 50 μm. i Bar graph showing the reduction of the BrdU-positive cells in the dorsal pallium of R26^Ddo/+ pups. j, m Representative images of clones generated by single E14 telencephalic progenitor cells transduced with retroviral vectors. Scale bar 100 µm. n Bar graph showing the average size of clones generated by the transduced progenitor cells (n = 6, total number of clones, Ctrl: 335, Ddo: 260). All data are expressed as mean ± SEM. **p < 0.01; ***p < 0.0001, compared with control group (Student’s t test).

Fig. 2. Ddo-overexpressing mice exhibit selective social recognition deficit.

a–k R26^Ddo/+ and R26^+/+ mice were tested in a series of behavioral tasks relevant to psychiatric phenotypes. a, b Analysis of grooming and rearing (duration, expressed in s; n = 8/genotype), (c) novelty-induced exploration test (traveled distance; expressed in cm, n = 15/genotype), (d) rotarod test (fall latency, expressed in s; n = 8/genotype), (e) open field test [time spent in the central area, expressed as percentage (%) of total time (peripheral + central area); n = 8/genotype], (f) elevated plus-maze test (time spent in the arms, expressed in s; n = 10/genotype), (g) marble burying test (expressed as number of buried murbles; n = 10/genotype), (h) prepulse inhibition of the startle reflex [expressed as percentage (%); n = 8/genotype], (i) social interaction (interaction time, expressed in s; n = 8/genotype), (j) sociability and (k) social novelty test (n = 14/genotype). j, k Sociability and social novelty tests were analyzed as both time spent sniffing (expressed as s, left graphs) and discrimination index [expressed as the ratio between the time spent exploring the novel stimulus and the total exploration time, according to the following formulas: (i) sociability test: (time spent with mouse)/(time spent with mouse + time spent with object); (ii) social novelty test: (time spent with novel mouse)/(time spent with novel mouse + time spent with familiar mouse), right graphs]. i–k Representative drawings of the social behavior tasks are shown above graphs. All data are expressed as mean ± SEM. *p < 0.05; **p < 0.01, compared with novel object; ***p < 0.0001, compared with familiar mouse (Fisher’s post hoc comparison).

Fig. 3. Identification of DDO gene duplication and single nucleotide variants.

a a-CGH profile of chromosome 6 from the Agilent 4X180K array revealed duplication in 6q21 of ~128 kb involving DDO. Results are interpreted as log2 ratio of test versus control (in case of no mutation log2(2/2) = 0, in case of duplication: log2(3/2) > 0.58). The patient (a) exhibits a duplication inherited from her father (deceased) (c), absent in the mother (b). The green and red dots represent the log2 fluorescence ratios of individual oligonucleotide probes on the microarrays. The red dots are indicative of the duplication segment on chromosome 6 and represent probes with positive log2 fluorescence ratios; the green dots represent probes with negative log2 fluorescence ratios. b Family trio pedigree reporting the DNA sequence variants identified in each family member by DDO sequencing analysis. The only variant found in the proband (c.80-60A>G) (a) is inherited by the mother (b). The mother (b) of the patient was found to carry a nonsense mutation (c.20G>A; p.Trp7Ter) not previously reported. c RT-PCR on proband for DDO gene. d DDO gene Sanger Sequencing electropherograms: the proband (a) is wild-type for the nonsense variant c.20G>A (p.Trp7Ter) as the father (c), the mother shows the variant in heterozygosity. The proband presents the variant c.80 + 60A>G in herozygosity as the father, while the mother is homozygous for the same variant. F forward strand, R reverse strand, REF reference sequence. The red large arrows and the rectangles indicate the exact position of each variant at sequence level.

Fig. 4. Determination of D-aspartate, L-aspartate and other amino acids content in blood serum sample of the patient.

a Overlaid HPLC chromatograms illustrating the specificity of D-aspartate (D-Asp) peaks obtained from blood serum samples. The identity of the peak corresponding to D-aspartate was verified by treating serum sample with hDDO (inset, green line) or by adding the external standard to serum sample (inset, red line). b–i Amount of (b) D-aspartate, (c) L-aspartate, (d) D-aspartate/total aspartate (D + L) ratio, (e) L-asparagine; (f) D-serine, (g) L-serine, (h) D-serine/total serine (D + L) ratio and (i) glycine in the serum of the patient with DDO gene duplication, compared to gender- and age-matched healthy subjects (n = 7).