Clinical characterization of a novel RAB39B nonstop mutation in a family with ASD and severe ID causing RAB39B downregulation and study of a Rab39b knock down mouse model

Abstract

Autism spectrum disorder (ASD) and intellectual disability (ID) often exist together in patients. The RAB39B gene has been reported to be mutated in ID patients with additional clinical features ranging from ASD, macrocephaly, seizures and/or early-onset parkinsonism. Here, we describe a novel RAB39B nonstop mutation [Xq28; c.640 T > C; p.(*214Glnext*21)] in a family with ASD, severe ID and poor motor coordination, and we assessed the pathogenicity of the mutation. A heterologous cell system and a Rab39b knockdown (KD) murine model, which mimic the nonstop mutation, were used to validate the deleterious effect of the RAB39B mutation. The mutation led to RAB39B protein instability, resulting in its increased degradation and consequent downregulation. Using a Rab39b KD mouse model, we demonstrated that the downregulation of RAB39B led to increased GluA2 lacking Ca2+-permeable AMPAR composition at the hippocampal neuronal surface and increased dendritic spine density that remained in an immature filopodia-like state. These phenotypes affected behavioural performance in a disease-specific manner. Rab39b KD mice revealed impaired social behaviour but intact social recognition. They also showed normal anxiety-like, exploratory and motivational behaviours but impaired working and associative memories. In conclusion, we found a novel RAB39B nonstop variant that segregated in a family with a clinical phenotype including ID, ASD and poor motor coordination. The pathogenicity of mutations causing the downregulation of RAB39B proteins, impacting AMPAR trafficking and dendritic spine morphogenesis, reinforced the idea that AMPAR modulation and dendritic spine assets could be considered hallmarks of neurodevelopmental disorders.

Figure 1

RAB39B ter214Q mutation. (A) Probands II-1 and II-2 at 6.6 and 4.7 years of age. (B) Pedigree of the family. Circle is female and squares are male. Opened symbols indicate unaffected male and blue indicates the presence of RAB39B mutation. Light red and light blue squares and yellow circle specify the type of the disability and/or disorder presented. (C) DNA sequence from I-1, II-1 and II-2. Arrows indicate the nonstop mutation changing the TAG stop codon in CAG. Sequence numbers refer to the human RAB39B cDNA (NM_171998). (D) Human RAB39B protein sequence. In green, the CLC geranyl-geranylation site and in italic bold red the 21 aa tail.

Figure 2

RAB39B ter214Q characterization. (A) Expression level of Rab39b transcript in COS7 cells transfected with pFLAG or pFLAG-RAB39B WT or pFLAG-RAB39B ter214Q (n = 3). Data are presented as relative mRNA level of Rab39b calculated as 2^{-ΔCt(Rab39b–H3)}. (B) Representative western blots of COS7 cells expressing pFLAG (1 KDa, no band) or pFLAG-RAB39B WT (24.63 KDa) or pFLAG-RAB39B ter214gln (27.11 KDa) (n = 4), CALNEXIN (90 KDa) are used as housekeeping gene. (C) Graph indicates RAB39B protein expression normalized on CALNEXIN. (D) Representative western blots of COS7 expressing pFLAG or pFLAG-RAB39b WT or pFLAG-RAB39B ter214gln, treated with MG132 or DMSO as control (n = 3). CALNEXIN was used as housekeeping gene. (E) Graph indicates RAB39B protein expression normalized on CALNEXIN. MWM: molecular weight marker of protein ladder. Data are presented as mean ± SEM. Student’s t-test was used in (B, D and E) and two-way ANOVA interaction and Student’s t-test was used in (C).^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 3

RAB39B ter214Q characterization. (A) Representative images of COS7 cells transfected with pFLAG-RAB39B WT or p-FLAG-RAB39B ter214Q. Scale bar is 50 μm. (B—E) Morphological analysis of transfected COS7 cells. (F) Representative TIRF and epifluorescence images of COS7 cells transfected with pFLAG-RAB39B or pFLAG-RAB39B ter214gln together with MYC-PICK1 and GFP-GluA2. Quantification of surface GluA2 related on total RAB39B. Scale bar, 20 μm. Data are presented as mean ± SEM.

Figure 4

Generation and characterization of Rab39b KD animal model. (A) Scheme of the structural organization of the Rab39b locus where exons are black boxes spaced out from one intron, and 3′- and 5’-UTR are white boxes, of the targeting vector and of the Rab39b recombinant locus for the generation of Rab39b Knock Down (KD) by homologous recombination. Neo is neomycine cassette. BamHI and NcoI, restriction enzymes used. S1 and S2, Southern blot probes. FOR, Lox2 and 3’UTR, PCR primers for Rab39b KD genotyping. (B) Southern blot shows the identification of 1D3 and 2A3 recombinant clones. The 18Kb fragment for the wild type (WT) allele or 5.4Kb band for the recombinant allele were detected by S1 probe after BamHI digestion; the 6.7 Kb band for the WT allele or 4.4 Kb band for the recombinant allele were detected by S2 probe after NcoI digestion. (C) Representative western blot and (D) quantification of RAB39B total protein expression in relative pixel intensity normalized on the intensity of the calnexin band (1D3 = 4, 2A3 = 3). (E) Representative image of Rab39b WT and KD littermate mice. Scale bar 2 cm. (F) Body weight in grams (n = 10). (G) Representative image of Rab39b WT and KD brains and (H) quantification in grams (n = 3). Data are presented as mean ± SEM. Student’s t-test was used in (D). ^*P < 0.5; ^**P < 0.01; ^***P < 0.001.

Figure 5

RAB39B down-regulation alters AMPAR-trafficking and spine maturation. (A) Representative images of immunofluorescence of GluA1, GluA2 and GluA3 surface labelling in 14DIV GFP-transduced Rab39b WT and KD primary hippocampal neurons. Graphs indicate the AMPAR subunits neuronal surface expression density (GluA1: WT = 8, KD = 14; GluA2 WT = 19, KD = 28; GluA3: WT = 14, KD = 25 images from a minimum of three experimental replicates), expressed in number of positive puncta/μm². Scale bar 10 μm. (B) Representative images of apical dendrites from CA1-hippocampal area of 30 days old Rab39b WT and KD (n = 3) hippocampal slices, stained with Golgi impregnation. Scale bar 5 μm (C) Quantification of spine density expressed in number of spine-heads/μm. (D) Quantification of partitioning of spine morphology along a dendrite expressed in % of type of spine/dendrite. Data are presented as mean ± SEM. Student’s t-test was used. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 6

Rab39b KD mice show cognitive impairments. (A) Pictogram of 3-chamber sociability test. (B) Time in chamber in seconds during the phase1, (C) time spent sniffing the stranger1 (S1) or the object (Obj) during phase2 and (D) time spent sniffing stranger1 (S1) and the stranger2 (S2) in phase3 of sociability test was reported. (E) Pictogram of eight-arm radial maze apparatus. (F) Number of errors and (G) number of correct arm choices before the first error during the 8-arms radial maze test were presented. Dashed line is the chance level performance of 5.5 correct successive arm visits. (H) Pictogram of spontaneous alternation apparatus. (I) Number of visits and (J) the % of correct alternations during spontaneous alternation task were showed. (K-M) Delay and (N-P) trace fear conditioning protocols. (K, N) % of freezing elicited by repeated exposure to the conditioned stimulus (CS) during the training session. Pictograms on the graphs represents (K) the delay fear conditioning protocol where the 15 s tone (CS, black box) is superimposed with the foot shock (lightning) for the last 2 s, and (N) the trace fear conditioning protocol where the CS and the foot shock are separated by 15 s trace (white box). (L, M, O, P) % of freezing during memory test, 24 h after training, (L, O) for context, and (M, P) for tone. Data are presented as mean ± SEM. Mann–Whitney U test was used for (A–D) and ANOVA for (E–P). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.s