Humanized substitutions of Vmat1 in mice alter amygdala-dependent behaviors associated with the evolution of anxiety

Abstract

The human vesicular monoamine transporter 1 (VMAT1) harbors unique substitutions (Asn136Thr/Ile) that affect monoamine uptake into synaptic vesicles. These substitutions are absent in all known mammals, suggesting their contributions to distinct aspects of human behavior modulated by monoaminergic transmissions, such as emotion and cognition. To directly test the impact of these human-specific mutations, we introduced the humanized residues into mouse Vmat1 via CRISPR/Cas9-mediated genome editing and examined changes at the behavioral, neurophysiological, and molecular levels. Behavioral tests revealed reduced anxiety-related traits of Vmat1 ^Ile mice, consistent with human studies, and electrophysiological recordings showed altered oscillatory activity in the amygdala under anxiogenic conditions. Transcriptome analyses further identified changes in gene expressions in the amygdala involved in neurodevelopment and emotional regulation, which may corroborate the observed phenotypes. This knock-in mouse model hence provides compelling evidence that the mutations affecting monoaminergic signaling and amygdala circuits have contributed to the evolution of human socio-emotional behaviors.

Keywords: Behavior genetics; Evolutionary biology; Molecular Genetics; Molecular mechanism of behavior.

Graphical abstract

Figure 1

An ultra-conserved residue in VMAT1 exhibits functional variants unique to humans (A) A phylogenetic tree constructed by multiple sequence alignment of the VMAT1 gene. Almost all genes across 236 vertebrate species retain asparagine (Asn) on the 136th residue, whereas humans are the only vertebrate species except for bicolor damselfish (Stegastes partitus; shown in yellow) with a unique polymorphism (Thr136Ile; rs1390938). A gap in the aligned sequence is shown in gray. Note that the phylogenetic relationship presented here is not necessarily consistent with the known species tree. (B) hVMAT1 and (C) mVMAT1 protein structure predicted by homology modeling and the effects of human-type mutagenesis (corresponding to 133Thr and 133Ile in mVMAT1). (Top) When introduced in silico, 133Ile, and not 133Thr, exhibits hydrophobic interactions (shown by green dotted lines) with surrounding sites, which likely influences the folding and/or stability of mVMAT1 protein. Blue, red, and orange dotted lines represent amide bonds, hydrogen bonds, and weak van der Waals interactions, respectively. (Bottom) Δ Vibrational entropy energy between WT (133Asn) and mutants. Amino acids are colored according to the vibrational entropy change conferred by the given mutation. Red represents a gain of flexibility and blue represents a rigidification of the structure. The 133Ile mutation leads to the increased flexibility of the first luminal loop, a receptor-like domain affecting the affinity of ligands.

Figure 2

Generation of the Vmat1-humanized mouse models by CRISPR/Cas9 genome editing (A) Targeting strategy for mVMAT1 133Asn humanization. The genetic configuration of the mouse Vmat1 gene is shown above. Exon 4 encoding 133Asn is enlarged, and the primers used for genotyping (Exon4_F and Exon4_R) are depicted. To replace the mouse 133Asn with 133Thr or 133Ile by CRISPR/Cas9-mediated genome engineering, a guide RNA with minimum off-target effects was designed. GGG (gray) represents the PAM sequence. In addition to 133Asn humanization, restriction enzyme recognition sites (EcoRI and FspI) were synonymously incorporated to avoid unwanted re-editing and to simplify genotyping. Sanger sequencing profiles of 133Asn/Asn (WT), 133Thr/Thr, 133Ile/Ile, and 133Thr/Ile are shown on the right. (B) PCR-RFLP assay, in which PCR products amplified using Exon4_F and Exon4_R were digested by EcoRI and FspI, respectively, could be used to distinguish four genotypes without sequencing.

Figure 3

Comprehensive behavioral tests reveal distinct behavioral changes in Vmat1^Thr/Thr and Vmat1^Ile mice, including reduced anxiety (A) In Crawley’s social interaction test, Vmat1^Thr/Thr mice preferred a familiar (previously exposed) mouse over a novel stranger. (B) Structural Equation Modeling (SEM) revealed the best fit model to explain the effects of the Vmat1 genotype on locomotor activity (Act) and anxiety (Anx). According to the model, Vmat1^Ile (Vmat1^Thr/Ile and Vmat1^Ile/Ile) significantly reduces anxiety-like behavior. The boxes and circles represent measured and latent variables, respectively. The paths represent causal relationships on which numerical values indicate standardized coefficient and the gray-scale intensity of the paths indicates statistical significance tested by t-tests. See STAR Methods for the detailed modeling procedure. (C) Vmat1^Ile mice exhibited lower anxiety than WT and Vmat1^Thr/Thr genotypes. Male, 10 to 19-week-old mice were used in the behavioral experiments shown here (see the detailed information in Table S3). For the composite anxiety score, a higher value indicates lower anxiety. Statistical significance was evaluated by paired t-test for (A), and by pair-wise t-test with FDR correction by the Benjamini–Hochberg method for (C). Interactive effects of genotype (Ile allele labeled by 1, and 0 otherwise) and cage place were also assessed by the generalized additive model with quasi-Poisson distribution in (A). †: 0.05 < P < 0.1, ∗0.01 < P < 0.05, ∗∗0.001 < P < 0.01, ∗∗∗P < 0.001.

Figure 4

Differentially expressed genes (DEGs) in the brain among Vmat1 genotypes and predicted co-expressing modules (A) The number of DEGs detected by pair-wise comparisons among the four genotypes. All DEGs were found in the amygdala (with none in the prefrontal cortex or striatum). (B) Correlations between individual DEG expression levels for the WT vs. and Vmat1^Ile/Ile comparison and composite anxiety scores from LD, EP, OF, and SI tests (see STAR Methods). Only genes with strong Spearman’s correlations (P < 0.1) are shown. The bands are 95% confidence intervals. (C) Network dendrogram from co-expression modules based on the expression data of all 47 regional brain samples. Each branch represents an individual gene, and the colors below represent the module, correlation (ρ) with the behavioral phenotype (locomotor activity and anxiety), and the relative expression level in the amygdala across genotypes. The samples with asterisks are from 10-month-old male mice with behavioral data and were used to calculate the correlations between expression levels and behavioral phenotypes, and the others are from four-month-old male mice. The M1 module (shown in turquoise), showing negative correlations with the anxiety score, exhibited significant overrepresentation of the DEGs detected between WT and Vmat1^Ile/Ile mice. (D) Protein-protein interaction networks among the genes in M1. The DEGs detected between WT and Vmat1^Ile/Ile mice are shown in red. The thickness of the line indicates the strength of data supports analyzed by STRING.

Figure 5

Wild-type and Vmat1^Thr/Thr mice, but not Vmat1^Ile/Ile mice, exhibit a reduction in amygdalar 4–7 Hz local field potential (LFP) power under anxiogenic conditions (A) An elevated plus maze (EP) test. (B) (Left) LFP recordings were simultaneously performed from the dorsomedial prefrontal cortex (dmPFC) and basolateral amygdala (BLA). (Right) Histological confirmation of electrode locations in the dmPFC and BLA. (C) Typical LFP signals from the dmPFC and BLA. (D) Spectrograms of dmPFC (left) and BLA (right) LFP power in (anxiogenic) open arms relative to closed arms. Data were averaged from all Vmat1^WT mice. The bar above indicates the 4–7 Hz band, showing pronounced decreases in LFP power in both regions. (E) Spectral Granger causality averaged over 20 dmPFC–BLA electrode pairs. (F) Comparison of dmPFC and BLA LFP 4–7 Hz power (z-scored) between open and closed arms. ∗P < 0.05, Mann–Whitney U test followed by Bonferroni correction. Each line represents one mouse.