Mutations in SLIT and NTRK-like family member 1 (SLITRK1) result in Tourette syndrome (TS). Patients with TS exhibit delayed bone maturation and increased fracture risk. To understand the role of SLITRK1 in bone homeostasis, we examined the skeletal phenotype of Slitrk1 null mice and investigated the mechanisms responsible for altered bone cell function. Slitrk1 null mice had thinner cortical bones due to decreased periosteal bone formation, while trabecular bone density remained unchanged. Slitrk1 was expressed within osteoblast-lineage cells. Consequently, deletion of Slitrk1 in osteoblasts impaired ex vivo differentiation capacity. Loss of SLITRK1 in osteoblast cells reduced levels of TAZ, a RUNX2 co-activator necessary for osteoblast differentiation. These findings provide evidence for a cell-autonomous role of SLITRK1 in periosteal osteoblasts regulating cortical bone homeostasis. Our data further demonstrate that periosteal-, endosteal-, and trabecular-bone homeostasis are controlled by different mechanisms and provide new insight into the skeletal manifestations of TS.

Abstract Neural leucine-rich-repeat (LRR) transmembrane superfamily proteins have emerged as pivotal players in neural health and pathology. Notably, proteins characterized by LRR domains with no other discernible extracellular motifs have been established as crucial for synaptogenesis and synaptic maintenance. Lrtm2, a member of this LRR-exclusive group, displays pronounced expression in the striatum, globus pallidus, and substantia nigra pars reticulata of mouse brains, with its protein levels rising during postnatal development. Past research indicated that Lrtm2-deficient mice presented with modified motor functions and fluctuations in dopamine metabolite levels within the striatum and associated regions. Our study aimed to elucidate Lrtm2's role in striatal medium spiny neurons. Our findings show alterations in the distribution of proteins pivotal for neurotransmitter regulation in the striatonigral GABAergic pathway of Lrtm2-deficient mice. These observations led us to hypothesize Lrtm2's involvement in macromolecular trafficking within striatal neurons. To further discern the molecular mechanisms by which Lrtm2 may regulate macromolecular transport, we verified the co-localization of Lrtm2 and its potential binding partners in vivo.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients’ SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3–/–) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

The Slitrk family consists of six synaptic adhesion molecules, some of which are associated with neuropsychiatric disorders. In this study, we aimed to investigate the physiological role of Slitrk4 by analyzing Slitrk4 knockout (KO) mice. The Slitrk4 protein was widely detected in the brain and was abundant in the olfactory bulb and amygdala. In a systematic behavioral analysis, male Slitrk4 KO mice exhibited an enhanced fear memory acquisition in a cued test for classical fear conditioning, and social behavior deficits in reciprocal social interaction tests. In an electrophysiological analysis using amygdala slices, Slitrk4 KO mice showed enhanced long-term potentiation in the thalamo-amygdala afferents and reduced feedback inhibition. In the molecular marker analysis of Slitrk4 KO brains, the number of calretinin (CR)-positive interneurons was decreased in the anterior part of the lateral amygdala nuclei at the adult stage. In in vitro experiments for neuronal differentiation, Slitrk4-deficient embryonic stem cells were defective in inducing GABAergic interneurons with an altered response to sonic hedgehog signaling activation that was involved in the generation of GABAergic interneuron subsets. These results indicate that Slitrk4 function is related to the development of inhibitory neurons in the fear memory circuit and would contribute to a better understanding of osttraumatic stress disorder, in which an altered expression of Slitrk4 has been reported.

SLITRK1 is a neuronal transmembrane protein with neurite development-and synaptic formation-controlling abilities. Several rare variants of SLITRK1 have been identified and implicated in the pathogenesis of Tourette’s syndrome, trichotillomania, and obsessive–compulsive disorder, which can be collectively referred to as obsessive–compulsive-spectrum disorders. Recent studies have reported a possible association between bipolar disorder and schizophrenia, including a revertant of modern human-specific amino acid residues. Although the mechanisms underlying SLITRK1-associated neuropsychiatric disorders are yet to be fully clarified, rodent studies may provide some noteworthy clues. Slitrk1-deficient mice show neonatal dysregulation of the noradrenergic system, and later, anxiety-like behaviors that can be attenuated by an alpha 2 noradrenergic receptor agonist. The noradrenergic abnormality is characterized by the excessive growth of noradrenergic fibers and increased noradrenaline content in the medial prefrontal cortex, concomitant with enlarged serotonergic varicosities. Slitrk1 has both cell-autonomous and cell-non-autonomous functions in controlling noradrenergic fiber development, and partly alters Sema3a-mediated neurite control. These findings suggest that transiently enhanced noradrenergic signaling during the neonatal stage could cause neuroplasticity associated with neuropsychiatric disorders. Studies adopting noradrenergic signal perturbation via pharmacological or genetic means support this hypothesis. Thus, Slitrk1 is a potential candidate genetic linkage between the neonatal noradrenergic signaling and the pathophysiology of neuropsychiatric disorders involving anxiety-like or depression-like behaviors.

SLITRK1 is an obsessive-compulsive disorder spectrum-disorders-associated gene that encodes a neuronal transmembrane protein. Here we show that SLITRK1 suppresses noradrenergic projections in the neonatal prefrontal cortex, and SLITRK1 functions are impaired by SLITRK1 mutations in patients with schizophrenia (S330A, a revertant of Homo sapiens-specific residue) and bipolar disorder (A444S). Slitrk1-KO newborns exhibit abnormal vocalizations, and their prefrontal cortices show excessive noradrenergic neurites and reduced Semaphorin3A expression, which suppresses noradrenergic neurite outgrowth in vitro. Slitrk1 can bind Dynamin1 and L1 family proteins (Neurofascin and L1CAM), as well as suppress Semaphorin3A-induced endocytosis. Neurofascin-binding kinetics is altered in S330A and A444S mutations. Consistent with the increased obsessive-compulsive disorder prevalence in males in childhood, the prefrontal cortex of male Slitrk1-KO newborns show increased noradrenaline levels, and serotonergic varicosity size. This study further elucidates the role of noradrenaline in controlling the development of the obsessive-compulsive disorder-related neural circuit.

The striatum is involved in action selection, and its disturbance can cause movement disorders. Here, we show that leucine-rich repeats and transmembrane domain 2 (Lrtm2) controls protein sorting in striatal projection systems, and its deficiency causes disturbances in monoamine dynamics and behavior. The Lrtm2 protein was broadly detected in the brain, but it was enhanced in the olfactory bulb and dorsal striatum. Immunostaining revealed a strong signal in striatal projection output, including GABAergic presynaptic boutons of the SNr. In subcellular fractionation, Lrtm2 was abundantly recovered in the synaptic plasma membrane fraction, synaptic vesicle fraction, and microsome fraction. Lrtm2 KO mice exhibited altered motor responses in both voluntary explorations and forced exercise. Dopamine metabolite content was decreased in the dorsal striatum and hypothalamus, and serotonin turnover increased in the dorsal striatum. The prefrontal cortex showed age-dependent changes in dopamine metabolites. The distribution of glutamate decarboxylase 67 (GAD67) protein and gamma-aminobutyric acid receptor type B receptor 1 (GABA B R1) protein was altered in the dorsal striatum. In cultured neurons, wild-type Lrtm2 protein enhanced axon trafficking of GAD67-GFP and GABA B R1-GFP whereas such activity was defective in sorting signal-abolished Lrtm2 mutant proteins. The topical expression of hemagglutinin-epitope-tag (HA)-Lrtm2 and a protein sorting signal abolished HA-Lrtm2 mutant differentially affected GABA B R1 protein distribution in the dorsal striatum. These results suggest that Lrtm2 is an essential component of striatal projection neurons, contributing to a better understanding of striatal pathophysiology.

SLITRK2 encodes a transmembrane protein that modulates neurite outgrowth and synaptic activities and is implicated in bipolar disorder. Here, we addressed its physiological roles in mice. In the brain, the Slitrk2 protein was strongly detected in the hippocampus, vestibulocerebellum, and precerebellar nuclei-the vestibular-cerebellar-brainstem neural network including pontine gray and tegmental reticular nucleus. Slitrk2 knockout (KO) mice exhibited increased locomotor activity in novel environments, antidepressant-like behaviors, enhanced vestibular function, and increased plasticity at mossy fiber-CA3 synapses with reduced sensitivity to serotonin. A serotonin metabolite was increased in the hippocampus and amygdala, and serotonergic neurons in the raphe nuclei were decreased in Slitrk2 KO mice. When KO mice were treatedwithmethylphenidate, lithium, or fluoxetine, the mood stabilizer lithium showed a genotype-dependent effect. Taken together, Slitrk2 deficiency causes aberrant neural network activity, synaptic integrity, vestibular function, and serotonergic function, providing molecular-neurophysiological insight into the brain dysregulation in bipolar disorders.

Culture models of the blood-brain barrier (BBB) are important research tools. Their role in the preclinical phase of drug development to estimate the permeability for potential neuropharmaceuticals is especially relevant. Since species differences in BBB transport systems exist, primate models are considered as predictive for drug transport to brain in humans. Based on our previous expertise we have developed and characterized a non-human primate co-culture BBB model using primary cultures of monkey brain endothelial cells, rat brain pericytes, and rat astrocytes. Monkey brain endothelial cells in the presence of both pericytes and astrocytes (EPA model) expressed enhanced barrier properties and increased levels of tight junction proteins occludin, claudin-5, and ZO-1. Co-culture conditions also elevated the expression of key BBB influx and efflux transporters, including glucose transporter-1, MFSD2A, ABCB1, and ABCG2. The correlation between the endothelial permeability coefficients of 10 well known drugs was higher (R-2 = 0.8788) when the monkey and rat BBB culture models were compared than when the monkey culture model was compared to mouse in vivo data (R-2 = 0.6619), hinting at transporter differences. The applicability of the new non-human primate model in drug discovery has been proven in several studies.

Receptor transporter protein family members (RTP1-4) are known to promote the trafficking of G-protein coupled receptor to the cell surface membrane. Recent studies have shown that RTP4 interacts with mu opioid receptor-delta opioid receptor (MOPr-DOPr) heteromers and this leads to increase of the MOPr-DOPr heteromer levels at the cell surface after MOPr stimulation. In this study we explored the regulation of RTP4 expression by MOPr stiumlation and its mechanism. We treated Neuro2a cells that express MOPr and DOPr with DAMGO (MOPr ligand) found that DAMGO stimulation induces significant increase in RTP4 mRNA levels. By using several inhibitors of signaling molecules, we find that DAMGO-induced increase in RTP4 expression can be blocked by pre-treatment with the receptor-selective antagonist, Gai inhibitor, transcription inhibitor, and MEK inhibitor, while PKC inhibitor did not affect. Interestingly, JAK inhibitor also exhibited trend to decrease the effect of DAMGO. Together, these results suggest the expression of RTP4 is upregulated by MOPr activation via G-protein, MEK and/or JAK mediated pathways, and this, in turn, is likely to enhance the cell surface expression of MOPr-DOPr heteromers.

Trans-regulation of G protein-coupled receptors (GPCRs) by leucine-rich repeat (LRR) transmembrane proteins has emerged as a novel type of synaptic molecular interaction in the last decade. Several studies on LRR–GPCR interactions have revealed their critical role in synapse formation and in establishing synaptic properties. Among them, LRR–GPCR interactions between extracellular LRR fibronectin domain-containing family proteins (Elfn1 and Elfn2) and metabotropic glutamate receptors (mGluRs) are particularly interesting as they can affect a broad range of synapses through the modulation of signaling by glutamate, the principal excitatory transmitter in the mammalian central nervous system (CNS). Elfn–mGluR interactions have been investigated in hippocampal, cortical, and retinal synapses. Postsynaptic Elfn1 in the hippocampus and cerebral cortex mediates the tonic regulation of excitatory input onto somatostatin-positive interneurons (INs) through recruitment of presynaptic mGluR7. In the retina, presynaptic Elfn1 binds to mGluR6 and is necessary for synapse formation between rod photoreceptor cells and rod-bipolar cells. The repertoire of binding partners for Elfn1 and Elfn2 includes all group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), and both Elfn1 and Elfn2 can alter mGluR-mediated signaling through trans-interaction. Importantly, both preclinical and clinical studies have provided support for the involvement of the Elfn1–mGluR7 interaction in attention-deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), and epilepsy. In fact, Elfn1–mGluR7-associated disorders may reflect the altered function of somatostatin-positive interneuron inhibitory neural circuits, the mesolimbic and nigrostriatal dopaminergic pathway, and habenular circuits, highlighting the need for further investigation into this interaction.

LRFN2 encodes a synaptic adhesion-like molecule that physically interacts with N-methyl-D-aspartate (NMDA) receptor 1 and its scaffold proteins. Previous studies in humans and mice have demonstrated its genetic association with neurodevelopmental disorders such as learning deficiency and autism. In this study, we showed that Lrfn2-deficient (KO) mice exhibit abnormalities of erythropoietic systems due to altered NMDA receptor function. In mature Lrfn2 KO male mice, peripheral blood tests showed multilineage abnormalities, including normocytic erythrocythemia, and reduced platelet volume. Colony forming unit assay using bone marrow cells revealed decreases in the counts of erythrocyte progenitors (CFU-E) as well as granulocytes and monocyte progenitors (CFU-GM). Whole bone marrow cell staining showed that serum erythropoietin (EPO) level was decreased and EPO receptor-like immunoreactivity was increased. Flow cytometry analysis of bone marrow cells revealed increased early erythroblast count and increased transferrin receptor expression in late erythroblasts. Further, we found that late erythroblasts in Lrfn2 KO exhibited defective NMDA receptor-mediated calcium influx, which was inhibited by the NMDA receptor antagonist MK801. These results indicate that Lrfn2 has biphasic roles in hematopoiesis and is associated with the functional integrity of NMDA receptors in hematopoietic cells. Furthermore, taken together with previous studies that showed the involvement of NMDA receptors in hematopoiesis, the results of this study indicate that Lrfn2 may regulate erythropoiesis through its regulatory activity on NMDA receptors.

Stroke-prone spontaneously hypertensive rat (SHRSP) is widely accepted as an animal model of hypertension and cerebrovascular disease. SHRSP occurs BBB dysfunction even if chronic hypertension and onset of stroke are not well-established. The properties of BBB are maintained by cross-talk between brain endothelial cells and surrounded adjacent cells, such as astrocytes and pericytes. To reveal the detailed mechanism underlay the BBB dysfunction of SHRSP, we constructed an in vitroBBB model using brain endothelial cells, pericytes, and astrocytes. Isolated brain capillary endothelial cells (BECs) from SHRSP showed leaky barrier function and altered composition of tight junction proteins (claudin-5 and occludin). The co-culture method of BECs and astrocytes indicated that SHRSP astrocytes had less ability to induce barrier function on BECs than did astrocytes derived from normotensive control rat (WKY/Izm). In comparison using the triple co-culture model, SHRSP model showed a weak barrier function than WKY model. These results suggest that BBB-relate cells in SHRSP have different properties and the defective interactions among these cells. This altered cross-talk may be related to occurrences of cerebrovascular diseases in SHRSP.

Sphingosine 1-phosphate (S1P) is a major bioactive lipid mediator in the vascular and immune system. Here, we have shown that inhibition of S1P signaling prevents blood–brain barrier (BBB) dysfunction after ischemia both in vitro and in vivo. In the in vitro BBB models, oxygen-glucose deprivation and reoxygenation (OGD/R) enhanced the expression of an S1P synthesizing enzyme (Sphk1) and S1P transporters (Abca1, Spns2), increasing S1P in culture media. Inhibitors of Sphk1 (SKI-II) or Abca1 (probucol) attenuated the decrease in transendothelial electrical resistance and the increase in permeability caused by OGD/R. In the middle cerebral artery occlusion and reperfusion (MCAO/R) model of mice, probucol administration after MCAO operation reduced the infarction area and vascular leakage, preserving the integrity of tight junction proteins. Furthermore, MCAO/R caused activation of STAT3, a downstream mediator of S1P signaling, which was suppressed by postoperative probucol administration. Accordingly, S1P activated STAT3, both in cultured vascular endothelial cells and pericytes, and STAT3 signaling inhibitor (Stattic) protected BBB dysfunction in OGD/R-treated in vitro BBB models. These results suggest that inhibition of S1P signaling is a strategy to treat BBB impairment after cerebral ischemia and highlight the potential alternative use of probucol, a classical anti-hyperlipidemic drug, for emergency treatment of stroke.

Blood-brain barrier (BBB) functions are maintained by cross-talk between brain capillary endothelial cells and elements of the neurovascular unit. Brain pericytes have crucial roles to maintain the BBB functions. Sphingosine-1-phosphate (S1P) is known as the regulator of many biological processes. The aim of the present study was to examine the role of S1P as interaction factors between endothelial cells and pericytes. To examine the effects of S1P on barrier functions, we made two kinds of in vitro BBB models, endothelial cells monolayer model and endothelium-pericytes co-cultured model. Barrier functions were assessed by measuring TEER and permeability of sodium fluorescein (NaF). S1P treatment decreased the TEER value and increased the NaF-permeability on endothelial monolayer model. The S1P-induced barrier dysfunctions were worsened by the presence of pericytes. To examine whether S1P affects the production of secreted factors from pericytes, real-time PCR was performed. S1P increased the several mRNA expression related to inflammation. These data indicate that secreted factors from pericytes stimulated by exogenous S1P worsen the BBB functions, and S1P act as the interplay factors in blood-brain barrier.

Zic family genes encode C2H2-type zinc finger proteins that act as critical toolkit proteins in the metazoan body plan establishment. In this study, we searched evolutionarily conserved domains (CDs) among 121 Zic protein sequences from 22 animal phyla and 40 classes, and addressed their evolutionary significance. The collected sequences included those from poriferans and orthonectids. We discovered seven new CDs, CD0-CD6, (in order from the N- to C-terminus) using the most conserved Zic protein sequences from Deuterostomia (Hemichordata and Cephalochordata), Lophotrochozoa (Cephalopoda and Brachiopoda), and Ecdysozoa (Chelicerata and Priapulida). Subsequently, we analyzed the evolutionary history of Zic CDs including the known CDs (ZOC, ZFD, ZFNC, and ZFCC). All Zic CDs are predicted to have existed in a bilaterian ancestor. During evolution, they have degenerated in a taxa-selective manner with significant correlations among CDs. The N terminal CD (CD0) was largely lost, but was observed in Brachiopoda, Priapulida, Hemichordata, Echinodermata, and Cephalochordata, and the C terminal CD (CD6) was highly conserved in conserved-type-Zic possessing taxa, but was truncated in vertebrate Zic gene paralogues (Zic1/2/3), generating a vertebrate-specific C-terminus critical for transcriptional regulation. ZOC was preferentially conserved in insects and in an anthozoan paralogue, and it was bound to the homeodomain transcription factor Msx in a phylogenetically conserved manner. Accordingly, the extent of divergence of Msx and Zic CDs from their respective bilaterian ancestors is strongly correlated. These results suggest that coordinated divergence among the toolkit CDs and among toolkit proteins is involved in the divergence of metazoan body plans.

Lrfn2/SALM1 is a PSD-95-interacting synapse adhesion molecule, and human LRFN2 is associated with learning disabilities. However its role in higher brain function and underlying mechanisms remain unknown. Here, we show that Lrfn2 knockout mice exhibit autism-like behavioural abnormalities, including social withdrawal, decreased vocal communications, increased stereotyped activities and prepulse inhibition deficits, together with enhanced learning and memory. In the hippocampus, the levels of synaptic PSD-95 and GluA1 are decreased. The synapses are structurally and functionally immature with spindle shaped spines, smaller postsynaptic densities, reduced AMPA/NMDA ratio, and enhanced LTP. In vitro experiments reveal that synaptic surface expression of AMPAR depends on the direct interaction between Lrfn2 and PSD-95. Furthermore, we detect functionally defective LRFN2 missense mutations in autism and schizophrenia patients. Together, these findings indicate that Lrfn2/LRFN2 serve as core components of excitatory synapse maturation and maintenance, and their dysfunction causes immature/silent synapses with pathophysiological state.

One of the causal genes for holoprosencephaly (HPE) is ZIC2 (HPE5). It belongs to the zinc finger protein of the cerebellum (Zic) family of genes that share a C2H2-type zinc finger domain, similar to the GLI family of genes. In order to clarify the role of Zic2 in gene regulation, we searched for its direct target genes using chromatin immunoprecipitation (ChIP). We identified TGIF1 (HPE4), another holoprosencephaly-causative gene in humans. We identified Zic2-binding sites (ZBS) on the 5′ flanking region of Tgif1 by in vitro DNA binding assays. ZBS were essential for Zic2-dependent transcriptional activation in reporter gene assays. Zic2 showed a higher affinity to ZBS than GLI-binding sequences. Zic2-binding to the cis-regulatory element near the Tgif1 promoter may be involved in the mechanism underlying forebrain development and incidences of HPE.

Lophotrochozoa is a sister taxon of Ecdysozoa in the Protostomia that includes mollusks, annelids, brachiopods, and platyhelminths. Recent studies have clarified the structure, expression, and roles of lophotrochozoan Zic family genes. Zic genes in oligochaete annelid Tubifex tubifex (freshwater sludge worm) and polychaete annelid Capitella teleta (bristle worm) are commonly expressed in a subset of developing brain and mesoderm derivatives. The latter includes the naïve mesoderm and the associated chaetal sacs in each body segment, although the segmentation processes differ between the two species. Furthermore, in brachiopod Terebratalia transversa (lamp shell), Zic is expressed in the anterior ectodermal domains and mesodermal derivatives, including those associated with the chaetal sacs. This result suggests the common involvement of Zic genes in the development of chaetae, a lophotrochozoan novelty acquired in the course of evolution. In addition, the highly simplified lophotrochozoan Dicyema acuticephalum (dicyemid mesozoan, a cephalopod endoparasite), which lost its gut, nervous system, and muscles during evolution, expresses its Zic genes in hermaphroditic gonads, highlighting the role of Zic genes in germ cell development. The role of Zic in head regeneration was revealed in studies on platyhelminth Schmidtea mediterranea (freshwater planarian). Planarian Zic expression was induced in a subpopulation of neoblasts that includes adult pluripotent stem cells. It is needed for head regeneration and production of an anterior signaling center. Suppression of Wnt-β-catenin signaling underlies Zic-mediated head regeneration, reminiscent of Wnt-β-catenin suppression by vertebrate Zic genes. Taken together, studies on the lophotrochozoan Zic genes are essential to understanding not only the roles of these genes in body plan evolution but also the molecular mechanism underlying adult stem cell regulation.