Oligodendrocyte dysfunction due to Chd8 mutation gives rise to behavioral deficits in mice.

Mutations in the gene encoding the chromatin remodeler CHD8 are strongly associated with autism spectrum disorder (ASD). CHD8 haploinsufficiency also results in autistic phenotypes in humans and mice. Although myelination defects have been observed in individuals with ASD, whether oligodendrocyte dysfunction is responsible for autistic phenotypes has remained unknown. Here we show that reduced expression of CHD8 in oligodendrocytes gives rise to abnormal behavioral phenotypes in mice. CHD8 was found to regulate the expression of many myelination-related genes and to be required for oligodendrocyte maturation and myelination. Ablation of Chd8 specifically in oligodendrocytes of mice impaired myelination, slowed action potential propagation and resulted in behavioral deficits including increased social interaction and anxiety-like behavior, with similar effects being apparent in Chd8 heterozygous mutant mice. Our results thus indicate that CHD8 is essential for myelination and that dysfunction of oligodendrocytes as a result of CHD8 haploinsufficiency gives rise to several neuropsychiatric phenotypes.

Labelling and optical erasure of synaptic memory traces in the motor cortex.

Dendritic spines are the major loci of synaptic plasticity and are considered as possible structural correlates of memory. Nonetheless, systematic manipulation of specific subsets of spines in the cortex has been unattainable, and thus, the link between spines and memory has been correlational. We developed a novel synaptic optoprobe, AS-PaRac1 (activated synapse targeting photoactivatable Rac1), that can label recently potentiated spines specifically, and induce the selective shrinkage of AS-PaRac1-containing spines. In vivo imaging of AS-PaRac1 revealed that a motor learning task induced substantial synaptic remodelling in a small subset of neurons. The acquired motor learning was disrupted by the optical shrinkage of the potentiated spines, whereas it was not affected by the identical manipulation of spines evoked by a distinct motor task in the same cortical region. Taken together, our results demonstrate that a newly acquired motor skill depends on the formation of a task-specific dense synaptic ensemble.

Synaptic functions and their disruption in schizophrenia: From clinical evidence to synaptic optogenetics in an animal model.

The adult human brain consists of approximately a hundred billion neurons, which are connected via synapses. The pattern and strength of the synaptic connections are constantly changing (synaptic plasticity), and these changes are considered to underlie learning, memory, and personality. Many psychiatric disorders have been related to disturbances in synaptogenesis and subsequent plasticity. In this review, we summarize findings of synaptic disturbance and its involvement in the pathogenesis and/or pathophysiology of psychiatric disorders. We will focus on schizophrenia, because this condition has a high proven heritability, which offers more unambiguous insights into the biological origins of not only schizophrenia but also related psychiatric disorders. To demonstrate the involvement of synaptopathy in psychiatric disorders, we discuss what knowledge is missing at the circuits level, and what new technologies are needed to achieve a comprehensive understanding of synaptopathy in psychiatric disorders.

Near-Infrared Optogenetic Genome Engineering Based on Photon-Upconversion Hydrogels.

Photon upconversion (UC) from near-infrared (NIR) light to visible light has enabled optogenetic manipulations in deep tissues. However, materials for NIR optogenetics have been limited to inorganic UC nanoparticles. Herein, NIR-light-triggered optogenetics using biocompatible, organic TTA-UC hydrogels is reported. To achieve triplet sensitization even in highly viscous hydrogel matrices, a NIR-absorbing complex is covalently linked with energy-pooling acceptor chromophores, which significantly elongates the donor triplet lifetime. The donor and acceptor are solubilized in hydrogels formed from biocompatible Pluronic F127 micelles, and heat treatment endows the excited triplets in the hydrogel with remarkable oxygen tolerance. Combined with photoactivatable Cre recombinase technology, NIR-light stimulation successfully performs genome engineering resulting in the formation of dendritic-spine-like structures of hippocampal neurons.

DISC1-dependent switch from progenitor proliferation to migration in the developing cortex.

Regulatory mechanisms governing the sequence from progenitor cell proliferation to neuronal migration during corticogenesis are poorly understood. Here we report that phosphorylation of DISC1, a major susceptibility factor for several mental disorders, acts as a molecular switch from maintaining proliferation of mitotic progenitor cells to activating migration of postmitotic neurons in mice. Unphosphorylated DISC1 regulates canonical Wnt signalling via an interaction with GSK3ß, whereas specific phosphorylation at serine 710 (S710) triggers the recruitment of Bardet-Biedl syndrome (BBS) proteins to the centrosome. In support of this model, loss of BBS1 leads to defects in migration, but not proliferation, whereas DISC1 knockdown leads to deficits in both. A phospho-dead mutant can only rescue proliferation, whereas a phospho-mimic mutant rescues exclusively migration defects. These data highlight a dual role for DISC1 in corticogenesis and indicate that phosphorylation of this protein at S710 activates a key developmental switch.

PAKs inhibitors ameliorate schizophrenia-associated dendritic spine deterioration in vitro and in vivo during late adolescence.

Drug discovery in psychiatry has been limited to chemical modifications of compounds originally discovered serendipitously. Therefore, more mechanism-oriented strategies of drug discovery for mental disorders are awaited. Schizophrenia is a devastating mental disorder with synaptic disconnectivity involved in its pathophysiology. Reduction in the dendritic spine density is a major alteration that has been reproducibly reported in the cerebral cortex of patients with schizophrenia. Disrupted-in-Schizophrenia-1 (DISC1), a factor that influences endophenotypes underlying schizophrenia and several other neuropsychiatric disorders, has a regulatory role in the postsynaptic density in association with the NMDA-type glutamate receptor, Kalirin-7, and Rac1. Prolonged knockdown of DISC1 leads to synaptic deterioration, reminiscent of the synaptic pathology of schizophrenia. Thus, we tested the effects of novel inhibitors to p21-activated kinases (PAKs), major targets of Rac1, on synaptic deterioration elicited by knockdown expression of DISC1. These compounds not only significantly ameliorated the synaptic deterioration triggered by DISC1 knockdown but also partially reversed the size of deteriorated synapses in culture. One of these PAK inhibitors prevented progressive synaptic deterioration in adolescence as shown by in vivo two-photon imaging and ameliorated a behavioral deficit in prepulse inhibition in adulthood in a DISC1 knockdown mouse model. The efficacy of PAK inhibitors may have implications in drug discovery for schizophrenia and related neuropsychiatric disorders in general.

Optogenetics: Applications in psychiatric research.

Recently, optogenetic techniques have emerged as a method to optically manipulate molecular and cellular events in target cells both in vitro and in vivo. Optogenetics results from the fruitful combination of optics and genetic engineering, maximizing the advantages of each discipline. These advantages are optical control through the manipulation of wavelength and light intensity on the millisecond timescale, and specific gene expression and gene product trafficking with subcellular precision. This kind of fine-tuning cannot be achieved using traditional methods. Therefore, optogenetic techniques have brought a revolution to neuroscience. In this review, we provide a concise summary of the history and recent advances of optogenetics, focusing in particular on applications for psychiatric research.

Sub-diffraction resolution pump-probe microscopy with shot-noise limited sensitivity using laser diodes.

We demonstrate the use of intensity-modulated laser diodes to implement pump-probe microscopy and achieved sub-diffraction resolution imaging with shot-noise limited sensitivity with a scheme of balanced detection. This technique has several applications for various types of induced transmission change, including excited-state absorption, ground state absorption bleaching and stimulated emission. By using our technique, biological imaging of mouse T cells and the axons of neurons in the cerebral cortex was demonstrated.

Disrupted-in-Schizophrenia-1 expression is regulated by beta-site amyloid precursor protein cleaving enzyme-1-neuregulin cascade.

Neuregulin-1 (NRG1) and Disrupted-in-Schizophrenia-1 (DISC1) are promising susceptibility factors for schizophrenia. Both are multifunctional proteins with roles in a variety of neurodevelopmental processes, including progenitor cell proliferation, migration, and differentiation. Here, we provide evidence linking these factors together in a single pathway, which is mediated by ErbB receptors and PI3K/Akt. We show that signaling by NRG1 and NRG2, but not NRG3, increase expression of an isoform of DISC1 in vitro. Receptors ErbB2 and ErbB3, but not ErbB4, are responsible for transducing this effect, and PI3K/Akt signaling is also required. In NRG1 knockout mice, this DISC1 isoform is selectively reduced during neurodevelopment. Furthermore, a similar decrease in DISC1 expression is seen in beta-site amyloid precursor protein cleaving enzyme-1 (BACE1) knockout mice, in which NRG1/Akt signaling is reportedly impaired. In contrast to neuronal DISC1 that was reported and characterized, expression of DISC1 in other types of cells in the brain has not been addressed. Here we demonstrate that DISC1, like NRG and ErbB proteins, is expressed in neurons, astrocytes, oligodendrocytes, microglia, and radial progenitors. These findings may connect NRG1, ErbBs, Akt, and DISC1 in a common pathway, which may regulate neurodevelopment and contribute to susceptibility to schizophrenia.

Effect of brain acidification on depression-related behaviors in diabetes mellitus.

Major depressive disorder (depression) is a leading cause of disability. The severity of depression is affected by many factors, one of which being comorbidity with diabetes mellitus (DM). The comorbidity of depression with DM is a major public health concern due to the high incidence of both conditions and their mutually exacerbating pathophysiology. However, the mechanisms by which DM exacerbates depression remain largely unknown, and elucidating these regulatory mechanisms would contribute to a significant unmet clinical need. We generated a comorbid mouse model of depression and DM (comorbid model), which was extensively compared with depression and DM models. Depressive and anhedonic phenotypes were more severe in the comorbid model. We thus concluded that the comorbid model recapitulated exacerbated depression-related behaviors comorbid with DM in clinic. RNA sequencing analysis of prefrontal cortex tissue revealed that the brain pH homeostasis gene set was one of the most affected in the comorbid model. Furthermore, brain pH negatively correlated with anhedonia-related behaviors in the depression and comorbid models. By contrast, these correlations were not detected in DM or control group, neither of which had been exposed to chronic stress. This suggested that the addition of reduced brain pH to stress-exposed conditions had synergistic and aversive effects on anhedonic phenotypes. Because brain pH was strongly correlated with brain lactate level, which correlated with blood glucose levels, these findings highlight the therapeutic importance of glycemic control not only for DM, but also for psychiatric problems in patients with depression comorbid with DM.

Distorted neurocomputation by a small number of extra-large spines in psychiatric disorders.

Human genetics strongly support the involvement of synaptopathy in psychiatric disorders. However, trans-scale causality linking synapse pathology to behavioral changes is lacking. To address this question, we examined the effects of synaptic inputs on dendrites, cells, and behaviors of mice with knockdown of SETD1A and DISC1, which are validated animal models of schizophrenia. Both models exhibited an overrepresentation of extra-large (XL) synapses, which evoked supralinear dendritic and somatic integration, resulting in increased neuronal firing. The probability of XL spines correlated negatively with working memory, and the optical prevention of XL spine generation restored working memory impairment. Furthermore, XL synapses were more abundant in the postmortem brains of patients with schizophrenia than in those of matched controls. Our findings suggest that working memory performance, a pivotal aspect of psychiatric symptoms, is shaped by distorted dendritic and somatic integration via XL spines.

Glutathione is a physiologic reservoir of neuronal glutamate.

Glutamate, the principal excitatory neurotransmitter of the brain, participates in a multitude of physiologic and pathologic processes, including learning and memory. Glutathione, a tripeptide composed of the amino acids glutamate, cysteine, and glycine, serves important cofactor roles in antioxidant defense and drug detoxification, but glutathione deficits occur in multiple neuropsychiatric disorders. Glutathione synthesis and metabolism are governed by a cycle of enzymes, the γ-glutamyl cycle, which can achieve intracellular glutathione concentrations of 1-10mM. Because of the considerable quantity of brain glutathione and its rapid turnover, we hypothesized that glutathione may serve as a reservoir of neural glutamate. We quantified glutamate in HT22 hippocampal neurons, PC12 cells and primary cortical neurons after treatment with molecular inhibitors targeting three different enzymes of the glutathione metabolic cycle. Inhibiting 5-oxoprolinase and γ-glutamyl transferase, enzymes that liberate glutamate from glutathione, leads to decreases in glutamate. In contrast, inhibition of γ-glutamyl cysteine ligase, which uses glutamate to synthesize glutathione, results in substantial glutamate accumulation. Increased glutamate levels following inhibition of glutathione synthesis temporally precede later effects upon oxidative stress.

Gene expression analysis in lymphoblastoid cells as a potential biomarker of bipolar disorder.

Although there is an urgent need for biological diagnosis of bipolar disorder (BD), there have been no established biomarkers. Gene expression analysis in lymphoblastoid cells (LCLs) would be a promising candidate for biomarkers. In this study, 17 candidate genes were measured in the LCLs of patients with BD. Using the data of the first set of samples (13 patients with bipolar I disorder and 21 controls), three genes, ANK3, RASGRP1 and POLG1, were selected by the logistic regression analysis with a stepwise method. Using the discriminant function generated by this analysis, the first sample was discriminated with the sensitivity of 76% and specificity of 85%. By applying the same function to the second sample set (18 patients with bipolar I and 37 controls), bipolar I disorder could be discriminated from the controls with the sensitivity of 44% and specificity of 81% (χ(2)=3.97, P=0.046). This study was the first to suggest a possible role of gene expression analysis of ANK3, RASGRP1 and POLG1 in the LCLs as potential biomarkers of BD.

[Constructive Understanding of Multi-scale Dynamism of Neuropsychiatric Disorders].

Although synaptic abnormalities are implicated in the pathophysiology of schizophrenia, it remains unclear whether the synaptic pathology is a casual mechanism that controls the behaviors, or whether it is merely a secondary consequence of the disorder. Chaotic behavior and fluctuations in the pathophysiology of schizophrenia also make it challenging to gain a better understanding of the disorder. In this study, we focused on the disorders in a constructive and multi-scale manner, we attempt to elucidate the causal relationships across the hierarchy between schizophrenia-related genes and disease-related behaviors.

Optical interrogation of multi-scale neuronal plasticity underlying behavioral learning.

Behavioral learning is driven by adaptive changes in the activation of behaviorally relevant neuronal ensembles. This learning-specific reorganization of neuronal circuits is correlated with activity-dependent modifications of synaptic dynamics. However, a definitive causal link remains to be established. How is synaptic plasticity distributed among circuits to eventually shape behavioral learning? A multi-scale understanding of the progressive plasticity is hindered by the lack of techniques for monitoring and manipulating these events. The current rise of synaptic optogenetics, especially combined with brain-wide circuit imaging, opens an entirely new avenue for studying causality at multiple scales. In this review, we summarize these technical achievements and discuss challenges in linking the plasticity across levels to elucidate the multi-scale mechanisms of learning.

Multi-Scale Understanding of NMDA Receptor Function in Schizophrenia.

Schizophrenia is a chronic and disabling psychiatric disorder characterized by disturbances of thought, cognition, and behavior. Despite massive research efforts to date, the etiology and pathophysiology of schizophrenia remain largely unknown. The difficulty of brain research is largely a result of complex interactions between contributory factors at different scales: susceptible gene variants (molecular scale), synaptopathies (synaptic, dendritic, and cell scales), and alterations in neuronal circuits (circuit scale), which together result in behavioral manifestations (individual scale). It is likely that each scale affects the others, from the microscale to the mesoscale to the macroscale, and vice versa. Thus, to consider the intricate complexity of schizophrenia across multiple layers, we introduce a multi-scale, hierarchical view of the nature of this disorder, focusing especially on N-methyl-D-aspartate-type glutamate receptors (NMDARs). The reason for placing emphasis on NMDAR is its clinical relevance to schizophrenia, as well as its diverse functions in neurons, including the robust supralinear synaptic integration provided by N-methyl-D-aspartate-type glutamate (NMDA) spikes and the Ca2+ permeability of the NMDAR, which facilitates synaptic plasticity via various calcium-dependent proteins. Here, we review recent evidence implicating NMDARs in the pathophysiology of schizophrenia from the multi-scale perspective. We also discuss recent advances from optical techniques, which provide a powerful tool for uncovering the mechanisms of NMDAR synaptic pathology and their relationships, with subsequent behavioral manifestations.

Investigation of betaine as a novel psychotherapeutic for schizophrenia.

BACKGROUND: Betaine is known to act against various biological stresses and its levels were reported to be decreased in schizophrenia patients. We aimed to test the role of betaine in schizophrenia pathophysiology, and to evaluate its potential as a novel psychotherapeutic. METHODS: Using Chdh (a gene for betaine synthesis)-deficient mice and betaine-supplemented inbred mice, we assessed the role of betaine in psychiatric pathophysiology, and its potential as a novel psychotherapeutic, by leveraging metabolomics, behavioral-, transcriptomics and DNA methylation analyses. FINDINGS: The Chdh-deficient mice revealed remnants of psychiatric behaviors along with schizophrenia-related molecular perturbations in the brain. Betaine supplementation elicited genetic background-dependent improvement in cognitive performance, and suppressed methamphetamine (MAP)-induced behavioral sensitization. Furthermore, betaine rectified the altered antioxidative and proinflammatory responses induced by MAP and in vitro phencyclidine (PCP) treatments. Betaine also showed a prophylactic effect on behavioral abnormality induced by PCP. Notably, betaine levels were decreased in the postmortem brains from schizophrenia, and a coexisting elevated carbonyl stress, a form of oxidative stress, demarcated a subset of schizophrenia with "betaine deficit-oxidative stress pathology". We revealed the decrease of betaine levels in glyoxylase 1 (GLO1)-deficient hiPSCs, which shows elevated carbonyl stress, and the efficacy of betaine in alleviating it, thus supporting a causal link between betaine and oxidative stress conditions. Furthermore, a CHDH variant, rs35518479, was identified as a cis-expression quantitative trait locus (QTL) for CHDH expression in postmortem brains from schizophrenia, allowing genotype-based stratification of schizophrenia patients for betaine efficacy. INTERPRETATION: The present study revealed the role of betaine in psychiatric pathophysiology and underscores the potential benefit of betaine in a subset of schizophrenia. FUND: This study was supported by the Strategic Research Program for Brain Sciences from AMED (Japan Agency for Medical Research and Development) under Grant Numbers JP18dm0107083 and JP19dm0107083 (TY), JP18dm0107129 (MM), JP18dm0107086 (YK), JP18dm0107107 (HY), JP18dm0107104 (AK) and JP19dm0107119 (KH), by the Grant-in-Aid for Scientific Research on Innovative Areas from the MEXT under Grant Numbers JP18H05435 (TY), JP18H05433 (AH.-T), JP18H05428 (AH.-T and TY), and JP16H06277 (HY), and by JSPS KAKENHI under Grant Number JP17H01574 (TY). In addition, this study was supported by the Collaborative Research Project of Brain Research Institute, Niigata University under Grant Numbers 2018-2809 (YK) and RIKEN Epigenetics Presidential Fund (100214-201801063606-340120) (TY).

[Optical Erasure of the Synaptic Ensemble that Underlies Learning and Memory].

Dentritic spines are small membrane protrusions. Their regulation is thought to be important for memory storage, but the links between dentric spines and memory have been largely correlational because of a luck of techniques for manipulating individual spines. To overcome this problem, we have developed a novel synaptic optoprobe, AS-PaRac1, which is unique not only because it specifically labels recently potentiated spines, but also because it becomes possible to selectively shrink spines containing AS-PaRac1. This indicates that AS-PaRac1 can be use to specifically visualize the recently "written spines" and that the erasure of these spines is possible upon excitation with blue light. Using in vivo two-photon imaging, synaptic potentiation was visualized during active remodeling of the neocortex. Upon learning a motor skill, AS-PaRac1 expression was induced in a relatively small number of neurons, in which approximately 8% of spines were tagged by AS-PaRac1. The labeled spines were broadly distributed throughout the dendritic tree. Excitation with blue light induced shrinkage of learning related spines and disrupted the acquired motor learning. In contrast, the erasure of a similar number of learning-irrelevant spines did not affect task performance. This novel light-dependent tool will open up new areas of memory research, and will additionally shed light on the neural networks that determine who we are.

Calcineurin knockout mice show a selective loss of small spines.

Calcineurin is required for long-term depression and activity-dependent spine shrinkage, and calcineurin mutations have been identified in patients with schizophrenia. Moreover, mice with conditional knockout of calcineurin B (CNB-KO) exhibit behavioral abnormalities suggestive of schizophrenia. Changes in the dendritic spines of these mice, however, have not been investigated. We therefore examined the dendritic spines of CNB-KO mice, and observed a significant reduction in small spines and an increase in large spines in the prefrontal and visual cortices. The effect of CNB-KO on the spine sizes was relatively moderate, possibly due to the presence of spontaneous fluctuations (dynamics) in the dendritic spines themselves. Thus, CNB-KO mice showed a spine phenotype similar to those recently reported in patients with schizophrenia.