Deficiency of the Synaptic Adhesion Protein Leucine-Rich Repeat Transmembrane Protein 4 Like 1 Affects Anxiety and Aggression in Zebrafish

Abstract

Aim: Leucine-rich repeat transmembrane proteins (LRRTMs) are synaptic adhesion proteins that regulate synapse development and function. They interact transsynaptically with presynaptic binding partners to promote presynaptic differentiation. Polymorphisms of LRRTM4, one of the four members of this protein family, have been linked to multiple neuropsychiatric disorders and childhood aggression, but the underlying mechanisms and physiological function of LRRTM4 during behavior are currently unclear.

Methods: To characterize the role of this gene for brain function, we combined a battery of behavioral assays with transcriptomic and metabolomic analyses, using zebrafish as a model system.

Results: Our findings revealed that lrrtm4l1, a brain-specific zebrafish orthologue of human LRRTM4, exhibits a brain region-specific expression pattern similar to humans, with strong expression in the dorsal telencephalon, a brain area critical for regulating emotional-affective and social behavior. lrrtm4l1^-/- zebrafish displayed heightened anxiety and reduced aggression, while locomotion and social behavior remained unaffected by the gene knockout. Transcriptomic analysis of the telencephalon identified over 100 differentially expressed genes between wild-type and mutant zebrafish and an enrichment of pathways related to synaptic plasticity and neuronal signaling. The brain metabolome of lrrtm4l1^-/- zebrafish showed multiple alterations, particularly in the dopaminergic and adenosinergic neurotransmitter systems.

Conclusion: These findings suggest that LRRTMs may have functions beyond their established role in excitatory synapse development, such as the regulation of neurotransmission and behavior. Targeting LRRTM4 therapeutically may thus be an interesting novel approach to alleviate excessive aggression or anxiety associated with a number of neuropsychiatric conditions.

Keywords: aggression; anxiety; leucine‐rich repeat transmembrane protein 4; neurotransmission; synaptic plasticity.

FIGURE 1

Lrrtm4l1 expression in the adult zebrafish brain. (a) Midsagittal section showing intense lrrtm4l1 expression (purple) in the telencephalon (Tel), inferior lobe (IL) and optic tectum (OT). (b–e) Coronal sections of the telencephalon (b, c), optic tectum (d), hypothalamus (e, f) and inferior lobe (g, h). Sections have been counterstained with thionine acetate for anatomical orientation. Scale bars are 1 mm in (a) and 100 μm in (b–h). The red line in the brain inserts indicates the sectioning plane. Cer, Cerebellum; CIL, central nucleus of the inferior lobe; CM, mamillary bodies; Dc, central zone of the dorsal telencephalon; DIL, diffuse nucleus of the inferior lobe; Dl, lateral zone of the dorsal telencephalon; Dm, Medial zone of the dorsal telencephalon; Hd, dorsal zone of the periventricular hypothalamus; Hv, ventral zone of the periventricular hypothalamus; IL, inferior lobe, MO, medulla oblongata; OT, optic tectum; PGZ, periventricular gray zone of optic tectum; Tel, telencephalon; TLa, torus lateralis; Vv, ventral zone of the ventral telencephalon. Schematic zebrafish brain inserts created by BioRender.com.

FIGURE 2

lrrtm4l1^−/− zebrafish display heightened anxiety. (a) Schematic representation of the lrrtm4l1 gene. The black line within exon 2 indicates the position of the nonsense mutation of the sa21708 zebrafish mutant line. Numbers represent bp positions. (b) Representative tracking heatmaps of lrrtm4l1^+/+ (WT) and lrrtm4l1^−/− (KO) zebrafish in the open field test. (c) KO zebrafish swim a shorter distance and (d) spend less time in the centre zone of the open field test. (e) KO animals show no difference in time spent immobile, but (f) display increased angular velocity. (g) Representative swimming trajectories of WT and KO zebrafish in the novel tank diving test. (h) KO zebrafish spend less time in the top zone and (i) enter the top zone of the test tank less frequently. n = 23‐28/group. Student's t test or Mann–Whitney U test. **p < 0.01;*p < 0.05 KO versus WT. Data are presented as mean ± SEM.

FIGURE 3

lrrtm4l1^−/− zebrafish are less aggressive. (a) Lrrtm4l1^−/− (KO) zebrafish were less aggressive than lrrtm4l1^+/+ (WT) zebrafish during the mirror‐induced aggression (MIA) assay. (b) The genotype did not alter the latency to approach the mirror and (c) entries into the mirror zone. (d) Representative swimming trajectories of WT and KO zebrafish in the MIA test. (e) Test setup of the corridor social interaction (SI) test showing a test fish approaching the stimulus shoal. The social interaction zone is virtually highlighted in yellow. (f) WT and KO zebrafish spend an equal amount of time in the SI zone and (g) enter the SI zone equally often. n = 22‐28/group. Student's t test or Mann–Whitney U test. *p < 0.05 KO versus WT. Data are presented as mean ± SEM.

FIGURE 4

Neurotranscriptomic effects of lrrtm4l1 deletion. (a) Principal component analysis plot of the 200 most variable genes after differential expression analysis between lrrtm4l1^−/− (KO) and lrrtm4l1^+/+ (WT) zebrafish. (b) Heatmap of differentially expressed genes (DEGs; padj < 0.05 and LFC > |0.58|) between KO and WT zebrafish. Hierarchical clustering of samples and genes reveals large differences between KO and WT, but similar transcriptional patterns within the two lines. (c) Volcano plot displaying the DEGs between KO and WT. Selected genes have been highlighted. Golden dots indicate genes upregulated in KO more than log fold change 0.58, blue dots represent genes downregulated in HAZ more than LFC—0.58 and black dots represent genes not passing these thresholds as indicated by vertical dotted lines. The horizontal dotted line indicates the significance threshold (padj 0.05) (d) Functional annotation clustering using DAVID pathway analysis revealed a significantly enriched cluster (enrichment score ≥ 1.3) related to semaphoring‐plexin signaling. (e) g:Profiler functional enrichment analysis revealed enriched KEGG pathways related to fatty acids and amino acids. n = 4/group.

FIGURE 5

Neurotransmitter and brain metabolome changes in lrrtm4l1 ^−/− zebrafish. Telencephalic levels of (a) homovanillinic acid, (b) adenosine, (c) serotonin and (d) melatonin as measured by targeted metabolomics in lrrtm4l1 ^−/− (Hom) and lrrtm4l1^+/+ (WT) zebrafish. n = 10/group. Student's t test. Data are presented as violin plots with the horizontal black line indicating the median. (e) Volcano plot displaying metabolites detected by untargeted metabolomics. Metabolites with adjusted p‐values below 0.05 and a log2fold change ≥ |1| were regarded as significant. (f) Spectral matching of detected metabolites. (g) Violin plot displaying telencephalic levels of a metabolite similar to methyl vanillate. n = 9–10/group.