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

(A) Schematic of major Trio isoforms present in the adult mouse brain, with locations of engineered neurodevelopmental disease (NDD)-associated Trio variants: K1431M autism spectrum disorder (ASD)-associated missense variant in the GEF1 DH domain; K1918X schizophrenia (SCZ)-associated nonsense variant that lies just before the GEF2 domain; and M2145T bipolar disorder (BPD)-associated missense variant in the GEF2 DH domain. (B) Representative sequencing chromatograms of wild-type (WT), +/K1431M, +/K1918X, and +/M2145T 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. Data are presented 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). Numbers of mice quantified per group are annotated inside the bar. (K) +/K1431M and +/K1918X male mice had decreased latency to fall off an accelerating rotarod compared to WT male mice. Two-way ANOVA with post-hoc Bonferroni MC test identified differences from WT for each individual trial (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Linear regressions identified differences from WT in slopes (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. (L) WT mice displayed normal social preference and spent more time with a stranger mouse (Str.) relative to an inanimate object (Obj.) in a three-chamber interaction test. Social interactions were impaired in +/K1431M male mice. (M) WT mice spent more time with a novel object (N) than with a familiar object (F). Novel object recognition was impaired in +/K1918X male mice. All data are presented as mean ±SEM. For (L-M), two-way ANOVA with post-hoc Bonferroni MC test identified differences from WT (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. (N) 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. One-way ANOVA with post-hoc Bonferroni MC test identified differences from WT (nsp<0.1, *p<0.05). n=19 WT; 10 +/K1431M; 15]+/K1918X; 9 +/M2145T male mice.

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 (+/K1918X 12.125 ±0.074 mm, p=0.0096; +/M2145T 12.012 ±0.152 mm, p=0.0007; vs WT 12.414 ±0.049 mm). (B) Brain weight is significantly reduced in P42 +/K1431M, +/K1918X, and +/M2145T males compared to WT (+/K1431M 0.379 ±0.004 g, p=0.0006; +/K1918X 0.345 ±0.004 g, p<0.0001; +/M2145T 0.381 ±0.005 g, p=0.0016; vs WT 0.399 ±0.004 g). (C) Body weight is increased in P42 +/K1431M and decreased in +/K1918X males (+/K1431M 22.911 ±0.382 g, p=0.0034; +/K1918X 20.700 ±0.338 g, p=0.0153; vs WT 21.745 ±0.224 g). (D) Head widths normalized to body weight of P42 +/K1431M male mice were reduced 11.8% compared to WT mice (0.520 ±0.008 mm/g vs WT 0.589 ±0.011 mm/g, p=0.0001). Head width-to-body weight ratios were calculated per individual mouse, with mouse number per group annotated within the bar. (E) Brain weights normalized to body weight of P42 +/K1431M and +/K1918X male mice were reduced 9.9% and 9.2%, resp., compared to WT mice (+/K1431M 0.0165 ±0.0004, p=0.0002; +/K1918X 0.0167 ±0.0004, p=0.0015; vs WT 0.0184 ±0.0004). Ratios were calculated by dividing the mean brain weight (B) by the mean body weight (C) of overlapping but non-identical populations of mice (see (B) and (C) for mouse number per group). For (A-E), data are presented as mean ±SEM; only male mice are shown here (data for females in Supp. Fig. 2). One-way ANOVA with post-hoc Bonferroni MC test identified differences from WT (nsp<0.1, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; n=number of mice per group, annotated within the bar). (F) Representative Nissl-stained image of a 30 μm coronal slice from a P42 WT brain. Magnification of the cortex (dotted black box) reveals cortical layers. (G) Total cross-sectional brain area of Nissl-stained coronal sections was reduced by ∼9% in P42 +/K1918X brains (43.21 ±0.577 mm2 vs WT 47.29 ±0.823 mm2, p=0.0045). (H) Total thickness of the cortex, in the region marked by the dotted black box in (F), is reduced by ∼8% in P42 +/K1918X brains (1.304 ±0.02262 mm vs WT 1.416 ±0.01417 mm, p=0.0021). For (G,H), data are presented as mean ±SEM. Ordinary one-way ANOVA with post-hoc Bonferroni MC test identified differences from WT (**p<0.01; n=number of mouse brains, annotated within the bar). (I) Representative maximum projection fluorescence image and corresponding dendritic arbor reconstruction of a motor cortex Layer 5 pyramidal neuron (M1 L5 PN) from a WT mouse. (J,K) Dendritic field size as measured by convex hull analysis of basal (J) and apical (K) dendrite arbor reconstructions of M1 L5 PNs of P42 Trio variant mice. +/K1918X trended toward smaller basal dendritic field size (0.1172 ±0.0078 mm2 vs WT 0.1368 ±0.0077 mm2, p=0.0933), and both +/K1918X and +/M2145T had significantly smaller apical dendritic field size (+/K1918X 0.5157 ±0.0169 mm2, p=0.0460; +/M2145T 0.4893 ±0.0285 mm2, p=0.0062; vs WT 0.6081 ±0.0319 mm2) compared to WT L5 PNsOrdinary one-way ANOVA with post-hoc Bonferroni MC test (B) identified differences from WT (nsp<0.1, *p<0.05, **p<0.01). (L-M) Sholl analysis revealed basal (L) and apical (M) 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 at each radius centered at the soma (*p<0.05, **p<0.01, ***p<0.001) For (J-M), n=23 neurons from 17 WT mice, 22 neurons from 15 +/K1431M mice, 20 neurons from 12 +/K1918X mice, 20 neurons from 14 +/M2145T mice.

Trio variants differentially impact synapse ultrastructure and synaptic vesicle distribution.

(A) Representative electron micrograph (EM) images 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. Scale bar = 0.5 μm. (B) Asymmetric synapses from all EM images of M1 L5 were quantified (from 18 fields of view per mouse, 5 mice per group). 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 decreased in M1 L5 synapses of +/K1918X and +/M2145T mice (+/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 M1 L5 synapses were unchanged from WT in all Trio variants. For (C-E), number of measurements per mouse across all 5 mice per genotype are denoted in the corresponding figure. (F) Synaptic vesicles (SVs) counted per 100 nm of active zone (AZ) length in M1 L5 as a function of distance from the AZ. +/M2145T showed an increase in the readily releasable pool (RRP) identified as docked SVs (15 nm from AZ; 1.23 ±0.05 vs. WT 0.90 ±0.05) and an 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). (G) Total releasable pool, calculated as the number of SVs at 15-150 nm from AZ per area of distribution (nm2). RRP 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. Ordinary one-way ANOVA with post-hoc Bonferroni MC test identified differences from WT (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). For (F,G), 35-50 synapses per mouse were analyzed.

Trio variant mice exhibit deficits in synaptic signaling and LTP.

(A,D) Representative traces of miniature excitatory AMPAR-, NMDAR-mediated mEPSCs and (G) inhibitory postsynaptic currents mIPSCs in M1 L5 pyramidal neurons of WT, +/K1431M, +/K1918X and +/M2145T mice. (B) AMPAR-mediated mEPSC amplitudes (I) were significantly increased in +/K1431M (16.67 ± 1.04 pA) and +/K1918X (14.71 ± 0.92 pA) slices, with no observed changes in +/M2145T slices (13.90 ± 1.16 pA) 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). (E, F) NMDAR mEPSC frequencies were reduced in +/K1431M (0.89 ± 0.12 1/s; vs WT 1.3324 ± 0.11 1/s; n=9-13 neurons from ≥ 5-7 mice per group) and showed a slight but significant increase in +/M2145T mice (1.68 ± 0.10 1/s). (H, I) GABA/GlyR mIPSC amplitudes were significantly increased in +/K1918X vs WT (23.69 ± 2.89 pA; vs 15.86 ± 1.56 pA, resp.), while frequencies were decreased in +/K1431M and +/M2145T (0.94 ± 0.14 1/s and 1.64 ± 0.19 1/s resp.; vs WT 2.44 ± 0.20; n=16-26 neurons from ≥6-8 mice per group). For (B-I), data are presented as mean ± SEM. One-way ANOVA with post-hoc Bonferroni test identified differences from WT (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). (J) Averaged representative traces of baseline and post-TBS eEPSCs currents in M1 L5 PNs of WT and Trio variant mice. (K) Normalized eEPSCs 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 the 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 differences from WT (****p<0.0001; n=6-8 neurons from ≥4-5 mice per group).

Trio GEF damaging variant mice have deficiencies in excitatory neurotransmitter release.

(A) Representative traces recorded from M1 L5 PNs of WT, Trio variant mice, and rescue experiment in acute slices of +/K1431M with 100 µM NSC23766 (NSC, Rac1 inhibitor) in response to paired-pulse stimulation in M1 L2/3. (B) Paired-pulse ratio (PPR) at varying interstimulus intervals (ISI), overlaid with a single exponential fit (except for +/M2145T data). An increase in the initial PPR was observed in M1 L5 PNs of +/K1431M mice (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 changes in +/K1918X. In +/M2145T slices, we observed a decrease in initial PPR at shorter ISI (35 ms: 1.05 ± 0.06, p<0.0001; 60 ms: 0.97 ± 0.06, p=0.037) and an increase at longer ISI (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). Acute application of NSC onto +/K1431M slices significantly shifted the PPR curve downwards at all points 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). (C) Representative traces of AMPAR eEPSCs in M1 L5 PNs under HFS (15 pulses at 40 Hz) in L2/3. (D) AMPAR eEPSCn amplitudes normalized to eEPSC1 of the train revealed changes in the depletion rates during HFS in Trio variants compared to WT (tau decay (τd), WT: 2.70 s, +/K1431M: 3.19 s, +/M2145T: 4.79 s, +/K1918X: 3.52 s, +/K1431M + NSC: 2.68 s; n=12-15 neurons from 5-7 mice). (E) The estimated glutamate probability of release (Pr) was decreased in +/K1431M (0.13 ± 0.099) and increased in +/M2145T (0.26 ± 0.019), with no significant change in +/K1918X (0.15 ± 0.01) compared to WT M1 L5 PNs (0.19 ± 0.01; n=11-15 neurons from ≥ 5 mice per group); acute NSC application rescued Pr in +/K1431M (0.22 ±0.019; n=11-15 neurons from ≥ 5 mice per group). (F) The calculated size of the readily releasable vesicle pool (RRP) was increased in +/M2145T M1 L5 PNs compared to WT (665.7 ± 68.5 pA vs 415.8 ± 43.9 pA). RRP in +/K1431M synapses before or after treatment with NSC did not differ from WT (543.1± 64.4 pA; + NSC: 427.9± 79.2 pA vs 415.8 ± 43.9 pA) (G) Exponential fits of the fractional recovery plotted vs ISI. Time of recovery, measured by exponential tau recovery (τR), was significantly decreased in +/K1431M M1 L5 PNs (6.3 s, vs WT 1.7 s). +/K1431M exhibited an inability to fully recover to initial levels after ISI 18 s, while NSC application has allowed for full recovery at 18s. It improved but did not fully rescue tau recovery time in +/K1431M (5.2 s). 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).

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 proteins quantified by mass spectrometry in P21 cortex (7362 proteins, n=4 mice per genotype), sorted by normalized enrichment score (NES). Pathways with a positive NES are upregulated compared to WT; pathways with a negative NES are downregulated compared to WT. [] denotes the gene set: [R] = Reactome, [WP] = WikiPathways, [K] = KEGG. (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). For a complete list of all enriched pathways identified by GSEA and SynGO, see Supp. Table 3. (D) Representative immunoblots for Munc18-1, synaptophysin (Syp), syntaxin-1a (Stx1a), and synaptotagmin3 (Syt3) in synaptosomes isolated from P42 cortex of WT and Trio variant mice. (E-H) Normalized intensity levels from immunoblots of select presynaptic proteins from P42 cortical synaptosomes. Munc18-1, Syp, and Syt3 are significantly increased in +/M2145T synaptosomes compared to WT; 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 14 WT, 10 +/K1431M, 9 +/K1918X, and 7 +/M2145T male mice). (I) Representative immunoblots for Kalirin, Tiam1, and VAV2 in P42 cortical RIPA lysates of WT and Trio variant mice. (J-L) Normalized intensity levels from immunoblots of select RhoGEFs from P42 cortical lysates. Kalirin levels are unchanged in all Trio variant cortex compared to WT; Tiam1 levels are increased ∼47% in +/K1431M and increased ∼45% in +/M2145T cortex compared 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).