Eye Movement Abnormalities in Major Depressive Disorder.

Background: Despite their high lifetime prevalence, major depressive disorder (MDD) is often difficult to diagnose, and there is a need for useful biomarkers for the diagnosis of MDD. Eye movements are considered a non-invasive potential biomarker for the diagnosis of psychiatric disorders such as schizophrenia. However, eye movement deficits in MDD remain unclear. Thus, we evaluated detailed eye movement measurements to validate its usefulness as a biomarker in MDD. Methods: Eye movements were recorded from 37 patients with MDD and 400 healthy controls (HCs) using the same system at five University hospitals. We administered free-viewing, fixation stability, and smooth pursuit tests, and obtained 35 eye movement measurements. We performed analyses of covariance with group as an independent variable and age as a covariate. In 4 out of 35 measurements with significant group-by-age interactions, we evaluated aging effects. Discriminant analysis and receiver operating characteristic (ROC) analysis were conducted. Results: In the free-viewing test, scanpath length was significantly shorter in MDD (p = 4.2 × 10-3). In the smooth pursuit test, duration of saccades was significantly shorter and peak saccade velocity was significantly lower in MDD (p = 3.7 × 10-3, p = 3.9 × 10-3, respectively). In the fixation stability test, there were no significant group differences. There were significant group differences in the older cohort, but not in the younger cohort, for the number of fixations, duration of fixation, number of saccades, and fixation density in the free-viewing test. A discriminant analysis using scanpath length in the free-viewing test and peak saccade velocity in the smooth pursuit showed MDD could be distinguished from HCs with 72.1% accuracy. In the ROC analysis, the area under the curve was 0.76 (standard error = 0.05, p = 1.2 × 10-7, 95% confidence interval = 0.67-0.85). Conclusion: These results suggest that detailed eye movement tests can assist in differentiating MDD from HCs, especially in older subjects.

Chromosome 22q11.2 deletion causes PERK-dependent vulnerability in dopaminergic neurons.

BACKGROUND: The chromosome 22q11.2 deletion is an extremely high risk genetic factor for various neuropsychiatric disorders; however, the 22q11.2 deletion-related brain pathology in humans at the cellular and molecular levels remains unclear. METHODS: We generated iPS cells from healthy controls (control group) and patients with 22q11.2 deletion (22DS group), and differentiated them into dopaminergic neurons. Semiquantitative proteomic analysis was performed to compare the two groups. Next, we conducted molecular, cell biological and pharmacological assays. FINDINGS: Semiquantitative proteomic analysis identified 'protein processing in the endoplasmic reticulum (ER)' as the most altered pathway in the 22DS group. In particular, we found a severe defect in protein kinase R-like endoplasmic reticulum kinase (PERK) expression and its activity in the 22DS group. The decreased PERK expression was also shown in the midbrain of a 22q11.2 deletion mouse model. The 22DS group showed characteristic phenotypes, including poor tolerance to ER stress, abnormal F-actin dynamics, and decrease in protein synthesis. Some of phenotypes were rescued by the pharmacological manipulation of PERK activity and phenocopied in PERK-deficient dopaminergic neurons. We lastly showed that DGCR14 was associated with reduction in PERK expression. INTERPRETATION: Our findings led us to conclude that the 22q11.2 deletion causes various vulnerabilities in dopaminergic neurons, dependent on PERK dysfunction. FUNDING: This study was supported by the AMED under grant nos JP20dm0107087, JP20dm0207075, JP20ak0101113, JP20dk0307081, and JP18dm0207004h0005; the MEXT KAKENHI under grant nos. 16K19760, 19K08015, 18H04040, and 18K19511; the Uehara Memorial Foundation under grant no. 201810122; and 2019 iPS Academia Japan Grant.

ARHGAP10, which encodes Rho GTPase-activating protein 10, is a novel gene for schizophrenia risk.

Schizophrenia (SCZ) is known to be a heritable disorder; however, its multifactorial nature has significantly hampered attempts to establish its pathogenesis. Therefore, in this study, we performed genome-wide copy-number variation (CNV) analysis of 2940 patients with SCZ and 2402 control subjects and identified a statistically significant association between SCZ and exonic CNVs in the ARHGAP10 gene. ARHGAP10 encodes a member of the RhoGAP superfamily of proteins that is involved in small GTPase signaling. This signaling pathway is one of the SCZ-associated pathways and may contribute to neural development and function. However, the ARHGAP10 gene is often confused with ARHGAP21, thus, the significance of ARHGAP10 in the molecular pathology of SCZ, including the expression profile of the ARHGAP10 protein, remains poorly understood. To address this issue, we focused on one patient identified to have both an exonic deletion and a missense variant (p.S490P) in ARHGAP10. The missense variant was found to be located in the RhoGAP domain and was determined to be relevant to the association between ARHGAP10 and the active form of RhoA. We evaluated ARHGAP10 protein expression in the brains of reporter mice and generated a mouse model to mimic the patient case. The model exhibited abnormal emotional behaviors, along with reduced spine density in the medial prefrontal cortex (mPFC). In addition, primary cultured neurons prepared from the mouse model brain exhibited immature neurites in vitro. Furthermore, we established induced pluripotent stem cells (iPSCs) from this patient, and differentiated them into tyrosine hydroxylase (TH)-positive neurons in order to analyze their morphological phenotypes. TH-positive neurons differentiated from the patient-derived iPSCs exhibited severe defects in both neurite length and branch number; these defects were restored by the addition of the Rho-kinase inhibitor, Y-27632. Collectively, our findings suggest that rare ARHGAP10 variants may be genetically and biologically associated with SCZ and indicate that Rho signaling represents a promising drug discovery target for SCZ treatment.

Cell body shape and directional movement stability in human-induced pluripotent stem cell-derived dopaminergic neurons.

Neuronal migration is necessary in the process of the formation of brain architecture. Recently, we demonstrated that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons exhibit directional migration in vitro. However, it remains unclear how the cell shape is involved in their migration. In this study, we performed live imaging analyses using human iPSC-derived dopaminergic neurons. Our automated method, which can automatically identify the cell body shape and the cell position at specific time points, revealed that healthy iPSC-derived dopaminergic neurons migrate according to their shape. This migration behavior was out of accord in neurons derived from iPSCs carrying an RELN deletion. Our findings provide a novel theory that cell body orientation is related to the stability of movement direction for human dopaminergic neurons, under the regulation of RELN.

Generation and analysis of novel Reln-deleted mouse model corresponding to exonic Reln deletion in schizophrenia.

AIM: A Japanese individual with schizophrenia harboring a novel exonic deletion in RELN was recently identified by genome-wide copy-number variation analysis. Thus, the present study aimed to generate and analyze a model mouse to clarify whether Reln deficiency is associated with the pathogenesis of schizophrenia. METHODS: A mouse line with a novel RELN exonic deletion (Reln-del) was established using the CRISPR/Cas9 method to elucidate the underlying molecular mechanism. Subsequently, general behavioral tests and histopathological examinations of the model mice were conducted and phenotypic analysis of the cerebellar granule cell migration was performed. RESULTS: The phenotype of homozygous Reln-del mice was similar to that of reeler mice with cerebellar atrophy, dysplasia of the cerebral layers, and abrogated protein levels of cerebral reelin. The expression of reelin in heterozygous Reln-del mice was approximately half of that in wild-type mice. Conversely, behavioral analyses in heterozygous Reln-del mice without cerebellar atrophy or dysplasia showed abnormal social novelty in the three-chamber social interaction test. In vitro reaggregation formation and neuronal migration were severely altered in the cerebellar cultures of homozygous Reln-del mice. CONCLUSION: The present results in novel Reln-del mice modeled after our patient with a novel exonic deletion in RELN are expected to contribute to the development of reelin-based therapies for schizophrenia.

Application of eye trackers for understanding mental disorders: Cases for schizophrenia and autism spectrum disorder.

Studies of eye movement have become an essential tool of basic neuroscience research. Measures of eye movement have been applied to higher brain functions such as cognition, social behavior, and higher-level decision-making. With the development of eye trackers, a growing body of research has described eye movements in relation to mental disorders, reporting that the basic oculomotor properties of patients with mental disorders differ from those of healthy controls. Using discrimination analysis, several independent research groups have used eye movements to differentiate patients with schizophrenia from a mixed population of patients and controls. Recently, in addition to traditional oculomotor measures, several new techniques have been applied to measure and analyze eye movement data. One research group investigated eye movements in relation to the risk of autism spectrum disorder several years prior to the emergence of verbal-behavioral abnormalities. Research on eye movement in humans in social communication is therefore considered important, but has not been well explored. Since eye movement patterns vary between patients with mental disorders and healthy controls, it is necessary to collect a large amount of eye movement data from various populations and age groups. The application of eye trackers in the clinical setting could contribute to the early treatment of mental disorders.

Comparative Analyses of Copy-Number Variation in Autism Spectrum Disorder and Schizophrenia Reveal Etiological Overlap and Biological Insights.

Compelling evidence in Caucasian populations suggests a role for copy-number variations (CNVs) in autism spectrum disorder (ASD) and schizophrenia (SCZ). We analyzed 1,108 ASD cases, 2,458 SCZ cases, and 2,095 controls in a Japanese population and confirmed an increased burden of rare exonic CNVs in both disorders. Clinically significant (or pathogenic) CNVs, including those at 29 loci common to both disorders, were found in about 8% of ASD and SCZ cases, which was significantly higher than in controls. Phenotypic analysis revealed an association between clinically significant CNVs and intellectual disability. Gene set analysis showed significant overlap of biological pathways in both disorders including oxidative stress response, lipid metabolism/modification, and genomic integrity. Finally, based on bioinformatics analysis, we identified multiple disease-relevant genes in eight well-known ASD/SCZ-associated CNV loci (e.g., 22q11.2, 3q29). Our findings suggest an etiological overlap of ASD and SCZ and provide biological insights into these disorders.

Abnormalities of eye movement are associated with work hours in schizophrenia.

Eye movement abnormalities have been reported in schizophrenia; however, their influences on everyday life remain unknown. From data on 69 subjects with schizophrenia and 246 healthy subjects, we found positive correlations between eye movement measures and work hours, which were only significant in subjects with schizophrenia. This relationship was also confirmed in a multi-site dataset including 118 subjects with schizophrenia and 280 healthy subjects. These findings further strengthen our understanding of eye movement abnormalities and their relevance in clinical recovery.

Single-cell trajectory analysis of human homogenous neurons carrying a rare RELN variant.

Reelin is a protein encoded by the RELN gene that controls neuronal migration in the developing brain. Human genetic studies suggest that rare RELN variants confer susceptibility to mental disorders such as schizophrenia. However, it remains unknown what effects rare RELN variants have on human neuronal cells. To this end, the analysis of human neuronal dynamics at the single-cell level is necessary. In this study, we generated human-induced pluripotent stem cells carrying a rare RELN variant (RELN-del) using targeted genome editing; cells were further differentiated into highly homogeneous dopaminergic neurons. Our results indicated that RELN-del triggered an impaired reelin signal and decreased the expression levels of genes relevant for cell movement in human neurons. Single-cell trajectory analysis revealed that control neurons possessed directional migration even in vitro, while RELN-del neurons demonstrated a wandering type of migration. We further confirmed these phenotypes in neurons derived from a patient carrying the congenital RELN-del. To our knowledge, this is the first report of the biological significance of a rare RELN variant in human neurons based on individual neuron dynamics. Collectively, our approach should be useful for studying reelin function and evaluating mental disorder susceptibility, focusing on individual human neuronal migration.

Copy-number variation in the pathogenesis of autism spectrum disorder.

Autism spectrum disorder is a neurodevelopmental disorder present in 1% of the population, characterized by impairments in reciprocal social interaction, communication deficits and restricted patterns of behavior. Approximately 10% of the autism spectrum disorder population is thought to have large chromosomal rearrangements. Copy-number variations (CNV) alter the genome structure either by duplication or deletion of a chromosomal region. The association between CNV and autism susceptibility has become more apparent through the use of methods based on comparative genomic hybridization in screening CNV. The nature of the high CNV rate in the human genome is partly explained by non-allelic homologous recombination between flanking repeated sequences derived from multiple copies of transposons or mobile genetic elements. There are hotspots for CNV in the human genome, such as 16p11.2 and 22q11.2. Genes involved in CNV are supposed to have copy-number dose-dependent effects on the behavior of affected individuals. Animal models give insight into the possible interactions between core genetic loci and additional factors contributing to the phenotypes of each individual. If affected genes code for cellular signaling molecules, reducing the dosage in the intracellular signaling pathway may result in the malfunction of the nervous system. The genetic background of autism spectrum disorder is highly heterogenic and most common or rare CNV do not lead to autism spectrum disorders in the majority of cases, but may occasionally increase the risk of developing an autism spectrum disorder.

[Autism spectrum disorder and genes for synaptic proteins].

Autism spectrum disorder (ASD) is characterized by impaired social interaction and communication, and restricted interests. It is generally accepted that ASD is caused by abnormalities in the structure or functions of the brain. Recent genome-wide analyses have identified copy number variations (CNVs) of neuronal genes in the genomes of ASD patients. CNV is a commonly observed phenomenon in human beings. During the first cell division of meiosis, irregular crossing over between homologous chromosomes results in loss or duplication of a segment. From 2007 to 2010, several groups performed a large-scale virtual screening of CNVs in ASD genomes. Genes affected by CNV, de novo CNVs, and rare CNVs were more prevalent in ASD. The results highlighted the CNVs of many neuronal genes associated with ASD. A fraction of these genes was previously identified in ASD but some were newly identified in each study. The CNVs implicated in ASD include neuronal genes belonging to 4 classes. These genes encode (1) neural adhesion molecules, including cadherins, neuroligin, and neurexin; (2) scaffold proteins such as SHANK3; (3) protein kinases and other intracellular signaling molecules; and (4) proteins that regulate protein syntheses. In general, these proteins play a role in synapse of glutamatergic neurons. The CNVs detected in the ASD patient genomes of imply a link between the synaptic proteins and pathological characteristics of ASD. Altered protein dosage by the CNVs may alter the functional quality of ASD patient's synapses, and may consequently affect their development of language and communication skills. There are 2 types of ASD, one is sporadic and, the other is familial. According to some reports, de novo CNVs are more frequently observed in sporadic-type ASD. However, it is generally understood that a combination of particular CNVs and other possible mutations underlie the pathology of ASD regardless of ASD type. The major symptoms of ASD are often curable with behavioral intervention during early childhood. An early diagnosis, followed by early start of treatment is crucial for language development and communication skills. Further and broader research on genomes will eventually provide information on the biological characteristics of ASD, as well as on specific ASD genotypes, thus aiding in the establishment of optimal treatment and medication to meet the biological conditions of each patient.

Retinal bipolar neurons express the cyclic nucleotide-gated channel of cone photoreceptors.

Cyclic nucleotide-gated (CNG) channels link intracellular cyclic nucleotides to changes in membrane ionic conductance in a variety of physiological contexts. In the retina, in addition to their central role in phototransduction, CNG channels may be involved in nitric oxide signaling in bipolar neurons or in the hyperpolarizing synaptic response to glutamate in ON-type (depolarizing) bipolar cells. Despite their potential physiological significance, however, expression of CNG channels has not yet been demonstrated in bipolar cells. To identify CNG channel subtypes in retinal bipolar neurons, we used single-cell molecular biological techniques in morphologically distinctive ON bipolar cells from goldfish retina. Both single-cell in situ hybridization and single-cell RT-PCR demonstrated in ON bipolar cells the presence of mRNA for the CNG channel subtype that is also found in cone photoreceptors. Other bipolar cells, which likely represent OFF cells, did not express the cone CNG channel. Thus the CNG channel of cone photoreceptors is expressed in ON bipolar cells, where it may be involved in physiological responses to nitric oxide, or in the sign-inverting glutamatergic synapse that gives rise to the ON visual pathway.

Drosophila regulatory factor X is necessary for ciliated sensory neuron differentiation.

Ciliated neurons play an important role in sensory perception in many animals. Modified cilia at dendrite endings serve as sites of sensory signal capture and transduction. We describe Drosophila mutations that affect the transcription factor RFX and genetic rescue experiments that demonstrate its central role in sensory cilium differentiation. Rfx mutant flies show defects in chemosensory and mechanosensory behaviors but have normal phototaxis, consistent with Rfx expression in ciliated sensory neurons and neuronal precursors but not in photoreceptors. The mutant behavioral phenotypes are correlated with abnormal function and structure of neuronal cilia, as shown by the loss of sensory transduction and by defects in ciliary morphology and ultrastructure. These results identify Rfx as an essential regulator of ciliated sensory neuron differentiation in Drosophila.