Heterozygosity for neurodevelopmental disorder-associated TRIO variants yields distinct deficits in behavior, neuronal development, and synaptic transmission in mice

Abstract

Genetic variants in TRIO are associated with neurodevelopmental disorders (NDDs) including schizophrenia (SCZ), autism spectrum disorder (ASD) and intellectual disability. TRIO uses its two guanine nucleotide exchange factor (GEF) domains to activate GTPases (GEF1: Rac1 and RhoG; GEF2: RhoA) that control neuronal development and connectivity. It remains unclear how discrete TRIO variants differentially impact these neurodevelopmental events. Here, we investigate how heterozygosity for NDD-associated Trio variants - +/K1431M (ASD), +/K1918X (SCZ), and +/M2145T (bipolar disorder, BPD) - impact mouse behavior, brain development, and synapse structure and function. Heterozygosity for different Trio variants impacts motor, social, and cognitive behaviors in distinct ways that model clinical phenotypes in humans. Trio variants differentially impact head and brain size, with corresponding changes in dendritic arbors of motor cortex layer 5 pyramidal neurons (M1 L5 PNs). Although neuronal structure was only modestly altered in the Trio variant heterozygotes, we observe significant changes in synaptic function and plasticity. We also identified distinct changes in glutamate synaptic release in +/K1431M and +/M2145T cortico-cortical synapses. The TRIO K1431M GEF1 domain has impaired ability to promote GTP exchange on Rac1, but +/K1431M mice exhibit increased Rac1 activity, associated with increased levels of the Rac1 GEF Tiam1. Acute Rac1 inhibition with NSC23766 rescued glutamate release deficits in +/K1431M variant cortex. Our work reveals that discrete NDD-associated Trio variants yield overlapping but distinct phenotypes in mice, demonstrates an essential role for Trio in presynaptic glutamate release, and underscores the importance of studying the impact of variant heterozygosity in vivo.

Fig. 1.. Genetically engineered mice with heterozygosity for K1431M, K1918X, or M2145T Trio variants have divergent effects on Trio protein expression and Rho GTPase activity.

(A) Schematic of major Trio isoforms present in the adult mouse brain, with locations of engineered neurodevelopmental disease (NDD)-associated Trio variants: K1431M is a rare missense variant in the GEF1 DH domain associated with autism spectrum disorder (ASD); a K1918X nonsense variant that lies just before the GEF2 domain associated with schizophrenia (SCZ); and M2145T missense variant in the GEF2 DH domain found in an individual with bipolar disorder (BPD). (B) Representative sequencing chromatograms of WT, and Trio variant mice. Arrows indicate heterozygosity for the variant alleles. (C) Representative immunoblots for Trio in P0 brain lysates using an antibody against Trio spectrin repeats (SR5–6). (D) Quantification of Trio protein levels from P0 brain lysates. Trio protein levels are reduced only in the brains of +/K1918X mice compared to WT controls (0.545 ± 0.126 of WT level, p=0.0046). (E-H) Activity levels of Rac1 (E,G) and RhoA (F,H) in whole brain homogenates of neonate (P0, E-F) and adult (P42, G-H) Trio variant mice as measured by G-LISA assay. Rac1 activity is increased in +/K1431M mice relative to WT at both ages (1.106 ± 0.027-fold at P0, p=0.0035; 1.509 ± 0.175-fold at P42, p=0.0279) and decreased in neonate +/K1918X mice (0.908 ± 0.0.032-fold, p=0.0230), with a trend towards increased activity in adult +/M2145T mice (1.438 ± 0.183-fold, p=0.0843); meanwhile RhoA activity appears unchanged in all mice relative to WT, though there may be a trend towards decreased activity in +/K1918X neonates (0.840 ± 0.074-fold, p=0.1292). (I,J) Activity levels of Rac1 (I) and RhoA (J) in synaptosomes isolated from P42 mouse cortex. Rac1 activity is increased in +/K1431M synaptosomes (1.125 ± 0.107-fold, p=0.0023), while RhoA activity is decreased in +/M2145T synaptosomes (0.731 ± 0.042-fold, p=0.0093) relative to WT. All data shown as mean ± SEM. For (D-J), one-way ANOVA with post-hoc Bonferroni MC test identified differences from WT (^nsp<0.1, *p<0.05, **p<0.01). Mouse numbers per group are shown in bars.

Fig. 2.. Heterozygosity for distinct Trio variants differentially impact NDD-like mouse behaviors.

(A) Schematic illustration of the behavioral tests performed on young adult (P42-P56) heterozygous Trio variant mice of both sexes. All mice proceeded through the same battery of tests. (B) +/K1431M and +/K1918X mice of both sexes had decreased latency to fall off an accelerating rotarod compared to WT male mice. In male mice (left), linear regressions identified differences from WT in slopes, indicating impaired rate of improvement in the skill (WT 16.96 ± 1.344; +/K1431M 7.270 ± 2.019, p<0.0001; +/K1918X 10.61 ± 1.444, p<0.0001; ^####p<0.0001) (n=40 WT; 10 +/K1431M; 16 +/K1918X; 13 +/M2145T male mice). In female mice (right), linear regressions identified differences from WT in slopes (+/K1431M 9.436 ± 2.146, p=0.0215; vs WT 14.52 ±1.792; ^#p<0.05) and intercepts (+/K1918X 6.492 ± 5.555, p=0.0248; vs WT 19.28 ± 5.942; ^€p<0.05) (n= 28 WT; 11 +/K1431M; 16 13 +/K1918X; and 15 +/M2145T female mice). (C) +/K1431M mice of both sexes and +/K1918X females showed impaired social interactions in a three-chamber test, showing no preference to the (Str.) vs. inanimate object (Obj.) compared to WT. (D) +/K1918X mice of both sexes and +/M2145T females exhibit impaired novel object recognition and spend equal time exploring a novel object (N) and a familiar object (F). (E) Male +/K1918X mice exhibited increased nestlet shredding over 30 min (26.26 ± 3.61% shredded vs WT 14.26 ± 2.97%; p=0.0433), and +/K1431M mice exhibited a trend toward increased nestlet shredding (25.90 ± 4.34% shredded, p=0.1038) compared to WT mice. n=19 male, 19 female WT; 10 male, 10 female +/K1431M; 15 male, 11 female +/K1918X; 9 male, 10 female +/M2145T mice. All data are shown as mean ± SEM, significant differences identified using two-way ANOVA with post-hoc Bonferroni MC (^nsp<0.1, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Numbers of mice quantified per group are annotated inside the bar unless otherwise indicated.

Fig. 3.. Trio +/K1431M and +/K1918X mice have smaller brain weights, but only +/K1918X brains have smaller less complex neurons.

(A) Ear-to-ear head width is reduced in P42 +/K1918X and +/M2145T compared to WT male mice (+/K1431M: 12.40 ± 0.04 mm, p=0.95; +/K1918X: 12.15 ± 0.08, p=0.001; +/M2145T 12.01 ± 0.15, p<0.0001; vs WT 12.49 ± 0.04 mm, n=17–45). (B) Brain weight is significantly decreased relative to WT in P42 males of all three heterozygous Trio variants (+/K1431M: 0.382 ± 0.004 g, p=0.04; +/K1918X: 0.346 ± 0.004 g, p<0.0001; +/M2145T 0.378 ± 0.005 g, p=0.002; vs WT 0.396 ± 0.004 g, n=44–98). (C) Body weight is significantly increased in P42 +/K1431M males and decreased in +/K1918X males (+/K1431M: 22.91 ± 0.38 g, p=0.01; +/K1918X: 20.67 ± 0.03 g, p=0.001; +/M2145T: 21.22 ± 0.33 g, p=0.44; vs WT 21.76 ± 0.19 g, n=45–118). (D) Head widths normalized to body weight of P42 +/K1431M male mice were reduced 10.8% compared to WT mice (+/K1431M: 0.520 ± 0.008 mm/g, p=0.0001; +/K1918X: 0.598 ± 0.012 mm/g, p>0.999; +/M2145T 0.607 ± 0.023 mm/g, p=0.54; vs WT 21.76 ± 0.19 mm/g, n=17–46). (E) Brain weights normalized to body weight of P42 +/K1431M and +/K1918X male mice were reduced 3.9% and 7.9%, respectively compared to WT mice (+/K1431M: 0.520 ± 0.008 mm/g, p=0.0001; +/K1918X: 0.598 ± 0.012 mm/g, p>0.999; +/M2145T 0.607 ± 0.023 mm/g, p=0.54; vs WT 21.76 ± 0.19 mm/g, n=17–46). (F) Representative images of Nissl-stained 30 μm coronal slices of male P42 WT and heterozygous Trio variant brains. (G) Total cross-sectional tissue area of Nissl-stained coronal sections were reduced ~9% +/K1918X in P42 male mice compared to WT. (H) Representative images of Nissl-stained cortical layers (L1-L6, dotted black box) of P42 WT and heterozygous Trio variant brains. (I) The total cortical thickness (from H) is reduced by ~8% in +/K1918X P42 male brains compared to WT. (J) Thickness of individual cortical layers, as identified in Nissl stains in Fig. 3H. L2/3 and L5 were preferentially reduced (−12% and −13%, resp.) in +/K1918X cortex relative to WT (L2/3: 0.306 ±0.011 mm vs WT 0.346 ±0.010 mm, p=0.0043; L5: 0.274 ±0.008 mm vs WT 0.314 ±0.008 mm, p=0.0054). (K) Representative traces of M1 L5 PNs from heterozygous male Trio variant mice crossed with Thy1-GFP(M). (L) +/K1918X M1 L5 PNs show a trend toward reduced basal dendritic field size (0.1172 ± 0.0078 mm²; vs WT 0.1368 ± 0.0077 mm², p=0.0933; n=15–22 neurons per mouse), as measured by convex hull analysis of dendrite arbor reconstructions. (M) Both +/K1918X and +/M2145T exhibit significantly smaller apical dendritic field size (+/K1918X: 0.5157 ± 0.0169 mm², p=0.0460; +/M2145T: 0.4893 ± 0.0285 mm², p=0.0062) compared to WT (0.6081 ± 0.0319 mm²; n=15–22 neurons per mouse). All data shown as mean ± SEM. One-way ANOVA with post-hoc Bonferroni MC test identified significant differences from WT (^nsp<0.1, *p<0.05, **p<0.01). (N,O) Sholl analysis revealed basal (N) and apical (O) dendritic arborization changes in Trio variant M1 L5 PNs compared to WT: both basal and apical arborization was reduced in +/K1918X, while proximal basal arborization was increased in +/K1431M. Two-way ANOVA (stacked) with post-hoc Bonferroni MC test identified differences from WT.

Fig. 4.. Trio variants differentially impact synapse ultrastructure and synaptic vesicle distribution.

(A) Representative electron micrographs (EMs) from motor cortex layer 5 (M1 L5) of P42 WT and Trio variant mice. Post-synaptic regions are pseudo-colored in cyan; pre-synaptic regions in magenta. (B) Asymmetric synapse density was increased in +/K1918X mice (0.09205 ± 0.004775 synapses/um²; vs WT 0.07633 ± 0.003954 synapses/um², p=0.0345). (C) PSD lengths were slightly decreased in M1 L5 synapses of by 6% in +/K1918X and 6.6% in +/M2145T mice vs WT (+/K1918X 0.2926 ± 0.004652 um, p=0.0204; +/M2145T 0.2916 ± 0.004922 um, p=0.0142; vs WT 0.3125 ± 0.005612 um). (D,E) Presynaptic bouton and spine head areas of Trio variants M1 L5 synapses were unchanged from WT. (F) Synaptic vesicles (SVs) distribution per 100 nm of active zone (AZ) length in M1 L5 as a function of distance from the AZ. +/M2145T showed an increase in readily releasable pool (RRP) identified as docked SVs (15 nm from AZ; 1.23 ± 0.05 vs WT 0.90 ± 0.05) and increase in tethered SVs (50 nm from AZ; 1.44 ± 0.04 vs WT 1.20 ± 0.05). +/K1918X and +/M2145T also showed an increase in the reserve pool of SVs (200 nm from AZ; 3.51 ± 0.21 and 3.81 ± 0.18, resp. vs WT 2.74 ± 0.16, n=15–30 synapses/mouse). (G) Total releasable pool, calculated as number of SVs at 15–150 nm from AZ per area of distribution (nm²). RRP (15–50 nm from AZ) was significantly increased in +/M2145T (0.257 ± 0.007 vs WT 0.228 ± 0.008), driven by increased docked and tethered SVs. All data are presented as mean ± SEM, significance tested by ordinary one-way ANOVA with post-hoc Bonferroni MC test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Fig. 5.. Trio variant mice exhibit deficits in synaptic signaling and LTP.

(A,D) Representative traces of miniature excitatory AMPAR-mediated mEPSCs, NMDAR-mediated mEPSCs, and (G) inhibitory postsynaptic currents (mIPSCs) in M1 L5 pyramidal neurons of WT and Trio variant mice. (B) AMPAR-mediated mEPSC amplitudes were significantly increased in +/K1431M (16.67 ± 1.04 pA; p=0.0009) and +/K1918X (14.71 ± 0.92 pA; p=0.03) slices, with no observed changes in +/M2145T slices (13.90 ± 1.16 pA; p=0.16) compared to WT (11.25 ± 0.84 pA; n=17–25 neurons from ≥6–8 mice per group). (C) No significant changes in AMPAR mEPSC frequencies (q) were observed in +/K1431M and +/K1918X, while +/M2145T had an increase (2.20 ± 0.15 1/s; vs WT 1.55 ± 0.09 1/s; p=0.0005). (E, F) NMDAR mEPSC frequencies were reduced in +/K1431M (0.89 ± 0.12 1/s; vs WT 1.3324 ± 0.11 1/s; p=0.015) and showed an increase in +/M2145T mice (1.68 ± 0.10 1/s vs WT 1.3324 ± 0.11 1/s; p=0.044, n=9–13 neurons from ≥5–7 mice per group). (H, I) GABA/GlyR mIPSC amplitudes were significantly increased in +/K1918X vs WT (23.69 ± 2.89 pA; vs 15.86 ± 1.56 pA, respectably; p=0.008), while frequency was decreased in +/K1431M and +/M2145T (0.94 ± 0.14 1/s, p<0.0001; and 1.64 ± 0.19 1/s, p=0.013; respectably; vs WT 2.44 ± 0.20; n=16–26 neurons from ≥6–8 mice per group). (J) Representative averaged traces of NMDA and AMPA eEPSCs recorded in M1 L5 PNs. (K) Heterozygous +/K1431M and +/K1918X Trio variants mice display reduced NMDAR/AMPAR eEPSC amplitude ratios, suggesting an increase in AMPAR transmission in M1 L5 PNs (+/K1431M: 0.75 ± 0.06, p=0.0002; +/K1918X: 0.69 ± 0.05, p<0.0001; +/M2145T: 1.00 ± 0.08, p=0.37; vs WT: 1.15 ± 0.07; n=13–19 neurons from ≥5–6 mice per group). (L) Averaged representative traces of baseline and post-TBS eEPSC currents in M1 L5 PNs of WT and Trio variant mice. (M) Normalized eEPSC amplitudes measuring LTP in L5 PNs by TBS in L2/3 afferents in all genotypes showed a significant decrease in the initiation and no potentiation of the LTP in +/K1431M and +/K1918X, with increase in initiation and potentiation of +/M2145T M1 L5 PNs compared to WT. LTP was induced at 0 min. RM two-way ANOVA with post-hoc Bonferroni MC test identified significant differences (n=6–8 neurons from ≥4–5 mice per group). Data are presented as mean ± SEM; significance tested by one-way ANOVA with post-hoc Bonferroni test unless specified otherwise (**p<0.01; ***p<0.001; ****p<0.0001).

Fig. 6.. Trio +/K1431M and +/M2145T variant mice have deficiencies in short-term facilitation, glutamate Pr and RRP.

(A) Representative traces in M1 L5 PNs of WT, Trio variant mice in response to paired pulse stimulation in L2/3. (B) Paired-pulse ratio (PPR) at varying interstimulus intervals (ISIs) were overlaid with a single exponential fit (except for +/M2145T data). An increase in the initial PPR was observed in M1 L5 PNs of +/K1431M slices (35 ms: 1.70 ± 0.089, p=0.003; 60 ms: 1.40 ± 0.07, p=0.046; 100 ms: 1.27 ± 0.05, p=0.031; n=20–34 neurons from ≥7–9 mice per group) with no change in +/K1918X slices; and in +/M2145T slices we observed a decrease in initial RRP at shorter ISIs (35 ms: 1.05 ± 0.06, p<0.0001; 60 ms: 0.97 ± 0.06, p=0.037) and an increase at longer ISIs (100 ms: 1.36 ± 0.09, p=0.034; 200 ms: 1.18 ± 0.08, p=0.013) compared to WT (35 ms: 1.40 ± 0.04; 60ms: 1.21 ± 0.03; 100 ms: 1.13 ± 0.03; 200 ms 1.0 ± 0.02; 300 ms 0.96 ± 0.17). (C) Representative traces of AMPAR eEPSCs in M1 L5 PNs under HFS (15 pulses at 40 Hz) in L2/3. (D) AMPAR eEPSC_n amplitudes normalized to eEPSC₁ of the train revealed changes in the depletion rates during HFS in Trio +/K1431M and +/M2145T variants compared to WT (tau decay (τ_d), WT: 2.7 s, +/K1431M: 3.19 s, +/M2145T: 4.79 s, +/K1918X: 2.52 s; n=12–15 neurons from 5–7 mice). (E) The estimated glutamate probability of release (Pr) was decreased in +/K1431M slices (0.13 ± 0.099; p=0.013) and increased in +/M2145T slices (0.26 ± 0.019, p=0.042), with no significant change in +/K1918X slices (0.15 ±0.01, p=0.64) compared to WT M1 L5 PNs (0.19 ± 0.01; n=12–15 neurons from ≥ 5 mice per group). (F) The calculated size of the readily releasable vesicle pool (RRP) was increased only in +/M2145T M1 L5 PNs compared to WT (665.7 ± 68.5 pA vs 415.8 ± 43.9 pA, p=0.012). RRP in +/K1431M and +/K1918X synapses did not differ from WT (543.1 ± 64.4 pA; and 543.1 ± 64.4 pA, respectively vs 415.8 ± 43.9 pA) (G) Exponential fits of the fractional recovery plotted vs ISI, to estimate synapse ability to recover from RRP depletion. Time of recovery, measured by exponential tau recovery (τ_R), was significantly decreased in +/K1431M M1 L5 PNs (5.7 s, vs WT 2.2 s), +/K1431M also exhibited an inability to fully recover to initial levels after ISI 18 s, vs WT. Data are presented as mean ± SEM, with significant differences from WT tested using one-way ANOVA with post-hoc Bonferroni (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Fig. 7. Trio variant mice show different molecular changes in the cortex involving presynaptic machinery and Rac1 GEFs.

(A) Bar graph illustrating the top enriched pathways (FDR q-value<0.2, *FDR<0.05) identified by gene set enrichment analysis (GSEA) for each Trio mutant mouse compared to WT, using all 7,362 proteins quantified by mass spectrometry in P21 cortex (n=4/genotype), sorted by normalized enrichment score (NES). Pathways with +NES are upregulated, -NES are downregulated vs. WT. [ ] indicates gene set: [R] Reactome, [WP] WikiPathways, [K] KEGG. Full list attached in Figure 7 – Source data 1 (B,C) Bar graphs illustrating the top enriched (FDR q-value <0.001) (B) cellular components and (C) biological processes identified by GSEA, using synaptic proteins from SynGO gene sets (n=1077 proteins), full list see in Figure 7 - Source data 2. (D) Representative immunoblots in synaptosomes isolated from P42 cortex of WT and Trio variant mice. (E-H) Normalized intensity levels from immunoblots demonstrate significant increases of (E) Munc18–1 (also known as syntaxin binding protein1), (F) synaptophysin (Syp), (G) syntaxin1a (Stx1) and (H) synaptotagmin3 (Syt3) levels in +/M2145T synaptosomes; Syp is increased while Stx1a is significantly decreased in +/K1431M synaptosomes compared to WT. Ordinary one-way ANOVA with post-hoc Bonferonni MC test identified differences from WT (*p<0.05, **p<0.01, ***p<0.001; n=synaptosomes from 7–14 male mice). (I) Representative immunoblots of select RhoGEFs from P42 cortical lysates of WT and Trio variant mice. (J-L) Normalized intensity levels from immunoblots identified ~47% increase of Tiam1 levels in +/K1431M and increase ~45% in +/M2145T cortex vs to WT; VAV2 is increased ~34% in +/M2145T cortex compared to WT. Unpaired t-tests identified differences from WT (*p<0.05; n=6 mice per genotype). Source data 1. Gene Set Enrichment Analysis of P21 cortex proteome of Trio WT, +/K1431M, +/K1918X, and +/M2145T mice. Source data 2. Synaptic Gene Ontologies (SynGO) of P21 cortex proteome of Trio WT, +/K1431M, +/K1918X, and +/M2145T mice.

Fig. 8.. NSC, Rac1 inhibitor application rescued Pr in +/K1431M L23-L5 synapses and improves SV recycling.

(A) Representative PPR traces of WT and Trio +/K1431M slices with or without 5 min application of 100 µM NSC23766. (B) Acute application of NSC onto both +/K1431M and WT synapses lead to decrease in PPF in M1 L2/3-L5 synapses. +/K1431M slices significantly shifted the PPF curve at all ISI downwards compared to untreated +/K1431M slices, and showed no significant difference from WT (+/K1431M +NSC 35 ms: 1.25 ± 0.06, p<0.0001; 60 ms: 1.13 ± 0.052, p=0.0007; 100 ms: 1.02 ± 0.053, p=0.0017; 200 ms 0.91 ± 0.039, p=0.0043; 300 ms 0.88 ± 0.045, p=0.021), with +/K1431M shifting into paired pulse depression at 200–300 ms intervals, while WT PPF plateauing to 1. (C) Representative traces of AMPAR eEPSCs in M1 L5 PNs under HFS of WT and Trio +/K1431M slices before and after NSC application. (D) Normalized AMPAR eEPSC_n amplitudes of the train revealed changes in the depletion rates during HFS before and after NSC application to WT and +/K1431M slices (tau decay (τ_d), WT+NSC: 2.85 s vs WT: 2.70 s; +/K1431M+NSC: 2.66 s vs +/K1431M: 3.19 s, n=12–15 neurons from 5–7 mice). (E) Rac1 inhibition by NSC increased the glutamate Pr in both WT and +/K1431M slices (WT + NSC 0.25 ± 0.067 vs initial 0.19 ± 0.01, p=0.046; and for +/K1431M + NSC 0.23 ± 0.019 vs initial 0.13 ± 0.099, p<0.0001; n=15–18 neurons from ≥ 5 mice per group). (F) RRP in WT or +/K1431M synapses with NSC did not show significant changes to initial values (WT +NSC: 370.3 ± 82.37 pA vs 415.8 ± 43.9 pA, p 0.99; +/K1431M + NSC: 427.9 ± 79.2 vs 543.1 ± 64.44 pA, p 0.99). (G) Exponential fits of the fractional recovery for WT and +/K1431M with and without NSC application. NSC application led to a faster recovery time in WT (+NSC: 1.5 s vs initial 2.2 s) and it significantly improved but did not fully rescue recovery time in +/K1431M (+ NSC 3.2 s vs initial 5.7 s), but allowed for full recovery at 18s. Data are presented as mean ± SEM, significance tested using one-way ANOVA with post-hoc Bonferroni (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).