Lack of Synaptic Adhesion Proteins Makes Zebrafish More Anxious and Less Aggressive

In this issue of Acta Physiologica, Tatzl et al. [1] investigate the relevance of the leucine-rich repeat transmembrane protein 4 like 1 (lrrtm4l1) gene, a zebrafish orthologue of the human LRRTM4, on behavioral, transcriptomic, and metabolomic readouts in mixed sex adult zebrafish (summarized in Figure 1). LRRTM4 is one of four members of the LRRTM gene family, which code for synaptic cell adhesion molecules that promote excitatory synapse development, including synaptic insertion and stabilization of AMPA receptors. LRRTM4 is associated with neurodevelopmental disorders, including autism spectrum disorder (ASD) and attempted suicide [2].

Using RNASeq, the authors identified 126 differentially expressed genes in the telencephalon of lrrtm4l1 knockout (lrrtm4l1^−/−) fish. Among these, they highlight the upregulation of rimkla and arhgap12. The rimkla gene is linked to cognition and memory consolidation [3]. The mammalian orthologue of the arhgap12b gene (i.e., ARHGAP12) promotes endocytosis of postsynaptic AMPA receptors, opposing the effect of LRRTM4 [4]. Notable downregulated genes include tyrosine hydroxylase, essential for catecholamine synthesis, and plasmolipin, encoding a major component of myelin sheaths; both genes have previously been linked to various disorders. Pathway analyses implicated semaphorin-plexin signaling, as well as fatty acid metabolism and degradation, as well as valine, leucine, and isoleucine degradation. Semaphorin-plexin signaling is involved in axon guidance and neuronal morphogenesis during neurodevelopment and might stabilize synaptic transmission both during development and adulthood [5].

Consistent with the differentially expressed genes, metabolomic changes in lrrtm4l1^−/− zebrafish revealed elevated levels of homovanillic acid, the end product of dopamine metabolism, and lower levels of adenosine. Additionally, lrrtm4l1^−/− zebrafish showed a trend toward higher serotonin and lower melatonin levels. Unbiased metabolite feature analysis revealed six significant features, including two upregulated features with fragmentation profiles similar to phospholipids and one downregulated feature that was similar to methyl vanillate.

To investigate the consequences of the observed transcriptomic and metabolic alterations, the authors performed several behavioral experiments in lrrtm4l1^−/− and wild-type zebrafish. In the open field test, lrrtm4l1^−/− fish displayed a lower total distance traveled and nominally increased immobile time. Additionally, their swimming movements appeared more erratic, and they displayed increased thigmotaxis, a potential indicator of increased anxiety-like behavior. The increased anxiety was further supported by increased bottom-dwelling in the novel tank diving test, as well as a reduced frequency of entries into the light zone of a light/dark tank (the light zone reflecting the more anxiogenic compartment), though the time spent in the light and dark compartments did not differ between genotypes. To test social (novelty) preference, the authors used the corridor social interaction test, where they found no differences between the two genotypes. Notably, despite the overall intact sociability, the lrrtm4l1^−/− fish displayed less aggressive behavior than their wild-type counterparts in the mirror test. In line with the reduced aggression, the authors could show high lrrtm4l1 expression levels in the telencephalic regions involved in the zebrafish social decision-making network (SDMN). The SDMN is activated by social encounters including aggression, possibly in a sex-specific manner [6].

In summary, transcriptomic and metabolic measures link lrrtm4l1 to fatty acid metabolism and degradation of certain amino acids, as well as neurodevelopment and synaptic transmission. The observed behavioral phenotype indicates a role of lrrtm4l1 in common psychiatric phenotypes. Future studies measuring neurotransmitters and gene expression in other brain regions could shed light on potential alterations of the catecholaminergic system in other regions, for example the diencephalon, in which dopaminergic neurons are prominent, as tyrosine hydroxylase was found downregulated and homovanillic acid levels increased. Additionally, as the authors suggest, testing for sex differences might provide further insights into the working mechanisms of lrrtm4l1, considering that the associated psychiatric phenotypes, that is, ASD and Tourette syndrome, are more commonly diagnosed in men than in women. Additionally, LRRTM4 showed a higher association with suicide attempt in women than in men [2], and aggressive behavior can also present differently between the sexes [6].

In a broader context, the findings from this study further support the relevance of disturbed excitatory synapse regulation in the pathology of psychiatric disorders with a potentially critical role of LRRTMs, particularly LRRTM4. In fact, excitatory (glutamatergic) signaling critically contributes to the pathophysiology of several psychiatric conditions including depression, schizophrenia, and neurodevelopmental disorders including ASD, and has been implicated in their treatment [7-9]. LRRTMs contribute to the development and plasticity of excitatory synapses by binding to their presynaptic partners, neurexins and heparan sulfate proteoglycans. Moreover, they contribute to the stabilization and maintenance of postsynaptic AMPA receptors (see schematic in Figure 1). Consistently, lack of LRRTM4 has been shown to reduce synaptic excitability and overall synapse number [10]. This likely explains the associations of LRRTMs with several mental disorders [2].

Furthermore, the study's findings showing upregulation of phospholipids and changes in fatty acid metabolism highlight an important role of lipid signaling in psychiatric disorders. To the best of our knowledge, this is the first study to associate LRRTMs with lipid signaling/metabolism. Lipids, lipid signaling, and lipid metabolism are increasingly recognized as important mediators of intra- and intercellular signaling in the brain. Changes in different lipid species and of genes involved in lipid metabolism and signaling have been found in a range of mental disorder patients and preclinical models. Importantly, lipid signaling has been linked to inflammation, which itself has been linked to psychiatric disorders. In fact, the consequences of inflammation on mental health have been suggested to be mediated via lipid metabolism, possibly by impairing signaling and plasticity of excitatory synapses [7, 11]. Thus, LRRTM4 might be an important mediator of the impact of inflammation and lipid signaling on synaptic pathologies.

Together, this study provides a compelling argument for further exploring LRRTM4 as a candidate target for neuropsychiatric interventions, especially in disorders marked by altered aggression and anxiety, but possibly also beyond.

Maja R. Adel: conceptualization, writing – original draft, writing – review and editing. Florian Freudenberg: conceptualization, writing – original draft, writing – review and editing, visualization.

The authors declare no conflicts of interest.