祖父江 顕

Tau hyperphosphorylation has been considered a major contributor to neurodegeneration in Alzheimer's disease (AD) and frontotemporal dementia, and related tauopathies have gained prominence in the development of therapies for these conditions. Glial responses are key features of AD and frontotemporal dementia, and are associated with neuroinflammation. Numerous transgenic mouse models that recapitulate critical AD-like pathology and cognitive impairment have been developed to examine pathogenic mechanisms and evaluate therapeutic approaches targeting tau and glial reactivity. Glial reactivity and neuroinflammation coincide with tau hyperphosphorylation, which induces behavioral impairment; however, the specific correlation between glial cell activation and abnormal behavior remains unknown. In this study, we investigated changes in glial cell gene expressions related to abnormal behaviors in rTg4510 mice, which phenocopy the tau pathology, neuroinflammation, and neurodegeneration observed in human tauopathies. Both 4- and 6-month-old rTg4510 mice displayed significantly impaired nest-building behavior compared with control mice. Paired association learning was also impaired in 4-month-old rTg4510 mice. Moreover, rTg4510 mice of both age groups exhibited abnormal exploratory behavior, and these mice spent a longer time in the open arms of the plus-maze test than control mice. Using a magnetic-activated cell-sorting technique, we analyzed glial cell gene expressions related to neuroinflammation, phagocytosis, and amyloid synthesis in the prefrontal cortex of rTg4510 mice. Regression analysis of glial gene expressions and behavioral tests revealed that various glial reactivities were associated with behavioral abnormalities. Our findings suggest specific genetic characteristics of glial cells that may lead to abnormal behavior in rTg4510 mice.

Systemic immune challenge can also cause neuropsychiatric abnormalities. Interferon-induced transmembrane protein 3 (IFITM3) plays a crucial role in cellular immune defense. Previously, we have demonstrated that IFITM3 affects neurodevelopment during the early developmental stage in mice, acting through innate immune activation. However, the pathophysiological significance of IFITM3 in immune system activation in adulthood remains unclear. To address this issue, we aimed to analyze the expression level of IFITM3 in the brain and the behavioral abnormalities in polyriboinosinic-polyribocytidylic acid (polyI:C)-treated adult male C57/BL6J wild-type (WT) and Ifitm3-/- mice. The expression levels of Ifitm3 mRNA and protein were significantly upregulated in the medial prefrontal cortex (mPFC), striatum, and hippocampus 24 h after polyI:C treatment in WT mice compared to saline-treated control mice. Furthermore, behavioral experiments revealed that polyI:C treatment induced cognitive dysfunction and anhedonia in WT mice, whereas Ifitm3-/- mice were resistant to these disorders. In conclusion, our results demonstrated that in adult mice, immune activation following polyI:C treatment may induce cognitive dysfunction and anhedonia through IFITM3 upregulation in the brain. These results suggest that IFITM3 is an attractive therapeutic target for neuropsychiatric dysfunction following immune activation in adulthood.

Alzheimer's disease (AD) is the leading neurodegenerative disease manifesting cognitive impairment. Its procession is regulated by activated glial cell-mediated neuroinflammation. Although estrogen deprivation is a known risk factor for AD in females, the impact of androgen deprivation on AD pathology in males, particularly regarding neuroinflammation, remains unclear. This study investigates the effects of long-term systemic androgen deprivation on AD pathology, including glial cell-specific gene expression, amyloid β (Aβ) pathology, and cognitive function in male castrated AppNL-G-F/NL-G-F (App) mice. We found significantly reduced androgen receptor (AR/Ar) expression levels in the precunei of male patients with early AD pathology and isolated brain microglia of male App mice compared with their nonpathological controls. In castrated App mice, microglial Tnf and Il6 and astrocytic Socs3 were downregulated, indicating that androgens may promote inflammation in the brain. However, Aβ accumulation and cognitive function were unaffected. These results suggest that although systemic androgen deprivation modulates neuroinflammation, the changes are insufficient to alter the AD phenotype or pathology in male App mice.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid β (Aβ) and phosphorylated tau. Neuroinflammation, mainly mediated by glial activation, plays an important role in AD progression. Although there is growing evidence for the anti-neuroinflammatory and neuroprotective effects of the cannabinoid system modulation, the detailed mechanism remains unclear. To address these issues, we analyzed the expression levels of cannabinoid receptor type II (Cnr2/Cb2) in AppNL-G-F/NL-G-F mice and human AD precuneus, which is vulnerable to amyloid deposition in AD, and the effects of JWH 133, a selective CB2 agonist, on neuroinflammation in primary glial cells and neuroinflammation and cognitive impairment in AppNL-G-F/NL-G-F mice. The levels of Cnr2/Cb2 were upregulated in microglia isolated from the cerebral cortex of AppNL-G-F/NL-G-F mice. CNR2 expression was also increased in RNAs derived from human precuneus with advanced AD pathology. Chronic oral administration of JWH 133 significantly ameliorated the cognitive impairment of AppNL-G-F/NL-G-F mice without neuropsychiatric side effects. Microglia and astrocyte mRNAs were directly isolated from the mouse cerebral cortex by magnetic-activated cell sorting, and the gene expression was determined by quantitative PCR. JWH 133 administration significantly decreased reactive astrocyte markers and microglial C1q, an inducer for the reactive astrocytes in AppNL-G-F/NL-G-F mice. In addition, JWH133 administration inhibited the expression of p-STAT3 (signal transducer and activator of transcription 3) in astrocytes in AppNL-G-F/NL-G-F mice. Furthermore, JWH 133 administration suppressed dystrophic presynaptic terminals surrounding amyloid plaques. In conclusion, stimulation of microglial CB2 ameliorates cognitive dysfunction in AppNL-G-F/NL-G-F mice by controlling astrocyte activation and inducing beneficial neuroinflammation, and our study has implications that CB2 may represent an attractive therapeutic target for the treatment of AD and perhaps other neurodegenerative diseases involving neuroinflammation.

Genetic factors significantly affect the pathogenesis of psychiatric disorders. However, the specific pathogenic mechanisms underlying these effects are not fully understood. Recent extensive genomic studies have implicated the protocadherin-related 15 (PCDH15) gene in the onset of psychiatric disorders, such as bipolar disorder (BD). To further investigate the pathogenesis of these psychiatric disorders, we developed a mouse model lacking Pcdh15. Notably, although PCDH15 is primarily identified as the causative gene of Usher syndrome, which presents with visual and auditory impairments, our mice with Pcdh15 homozygous deletion (Pcdh15-null) did not exhibit observable structural abnormalities in either the retina or the inner ear. The Pcdh15-null mice showed very high levels of spontaneous motor activity which was too disturbed to perform standard behavioral testing. However, the Pcdh15 heterozygous deletion mice (Pcdh15-het) exhibited enhanced spontaneous locomotor activity, reduced prepulse inhibition, and diminished cliff avoidance behavior. These observations agreed with the symptoms observed in patients with various psychiatric disorders and several mouse models of psychiatric diseases. Specifically, the hyperactivity may mirror the manic episodes in BD. To obtain a more physiological, long-term quantification of the hyperactive phenotype, we implanted nano tag® sensor chips in the animals, to enable the continuous monitoring of both activity and body temperature. During the light-off period, Pcdh15-null exhibited elevated activity and body temperature compared with wild-type (WT) mice. However, we observed a decreased body temperature during the light-on period. Comprehensive brain activity was visualized using c-Fos mapping, which was assessed during the activity and temperature peak and trough. There was a stark contrast between the distribution of c-Fos expression in Pcdh15-null and WT brains during both the light-on and light-off periods. These results provide valuable insights into the neural basis of the behavioral and thermal characteristics of Pcdh15-deletion mice. Therefore, Pcdh15-deletion mice can be a novel model for BD with mania and other psychiatric disorders, with a strong genetic component that satisfies both construct and surface validity.

BACKGROUND: Neuroinflammation substantially contributes to the pathology of Alzheimer's disease (AD), the most common form of dementia. Studies have reported that nuclear factor erythroid 2-related factor 2 (Nrf2) attenuates neuroinflammation in the mouse models of neurodegenerative diseases, however, the detailed mechanism remains unclear. METHODS: The effects of dimethyl fumarate (DMF), a clinically used drug to activate the Nrf2 pathway, on neuroinflammation were analyzed in primary astrocytes and AppNL-G-F (App-KI) mice. The cognitive function and behavior of DMF-administrated App-KI mice were evaluated. For the gene expression analysis, microglia and astrocytes were directly isolated from the mouse cerebral cortex by magnetic-activated cell sorting, followed by quantitative PCR. RESULTS: DMF treatment activated some Nrf2 target genes and inhibited the expression of proinflammatory markers in primary astrocytes. Moreover, chronic oral administration of DMF attenuated neuroinflammation, particularly in astrocytes, and reversed cognitive dysfunction presumably by activating the Nrf2-dependent pathway in App-KI mice. Furthermore, DMF administration inhibited the expression of STAT3/C3 and C3 receptor in astrocytes and microglia isolated from App-KI mice, respectively, suggesting that the astrocyte-microglia crosstalk is involved in neuroinflammation in mice with AD. CONCLUSION: The activation of astrocytic Nrf2 signaling confers neuroprotection in mice with AD by controlling neuroinflammation, particularly by regulating astrocytic C3-STAT3 signaling. Furthermore, our study has implications for the repositioning of DMF as a drug for AD treatment.

Recent single-cell analyses have revealed the complexity of microglial heterogeneity in brain development, aging, and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Disease-associated microglia (DAMs) have been identified in ALS mice model, but their role in ALS pathology remains unclear. The effect of genetic background variations on microglial heterogeneity and functions remains unknown. Herein, we established and analyzed two mice models of ALS with distinct genetic backgrounds of C57BL/6 and BALB/c. We observed that the change in genetic background from C57BL/6 to BALB/c affected microglial heterogeneity and ALS pathology and its progression, likely due to the defective induction of neurotrophic factor-secreting DAMs and impaired microglial survival. Single-cell analyses of ALS mice revealed new markers for each microglial subtype and a possible association between microglial heterogeneity and systemic immune environments. Thus, we highlighted the role of microglia in ALS pathology and importance of genetic background variations in modulating microglial functions.

The organelle contact site of the endoplasmic reticulum and mitochondria, known as the mitochondria-associated membrane (MAM), is a multifunctional microdomain in cellular homeostasis. We previously reported that MAM disruption is a common pathological feature in amyotrophic lateral sclerosis (ALS); however, the precise role of MAM in ALS was uncovered. Here, we show that the MAM is essential for TANK-binding kinase 1 (TBK1) activation under proteostatic stress conditions. A MAM-specific E3 ubiquitin ligase, autocrine motility factor receptor, ubiquitinated nascent proteins to activate TBK1 at the MAM, which results in ribosomal protein degradation. MAM or TBK1 deficiency under proteostatic stress conditions resulted in increased cellular vulnerability in vitro and motor impairment in vivo. Thus, MAM disruption exacerbates proteostatic stress via TBK1 inactivation in ALS. Our study has revealed a proteostatic mechanism mediated by the MAM-TBK1 axis, highlighting the physiological importance of the organelle contact sites.

The cytoplasmic aggregation of TAR DNA binding protein-43 (TDP-43), also known as TDP-43 pathology, is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism underlying TDP-43 cytoplasmic mislocalization and subsequent aggregation remains unclear. Here, we show that TDP-43 dimerization/multimerization is impaired in the postmortem brains and spinal cords of patients with sporadic ALS and that N-terminal dimerization–deficient TDP-43 consists of pathological inclusion bodies in ALS motor neurons. Expression of N-terminal dimerization–deficient mutant TDP-43 in Neuro2a cells and induced pluripotent stem cell–derived motor neurons recapitulates TDP-43 pathology, such as Nxf1-dependent cytoplasmic mislocalization and aggregate formation, which induces seeding effects. Furthermore, TDP-DiLuc, a bimolecular luminescence complementation reporter assay, could detect decreased N-terminal dimerization of TDP-43 before TDP-43 pathological changes caused by the transcription inhibition linked to aberrant RNA metabolism in ALS. These findings identified TDP-43 monomerization as a critical determinant inducing TDP-43 pathology in ALS.

Alzheimer's disease (AD) is the most common form of dementia, pathologically characterized by senile plaques and neurofibrillary tangles (NFTs), resulting in neurodegeneration. Neuroinflammation, defined as the activation of glial cells such as microglia and astrocytes, is observed surrounding senile plaques and affected neurons in AD. Recently conducted genome-wide association studies (GWAS) indicate that a large section of identified AD risk genes are involved in immune responses and are enriched in microglia. Microglia are innate immune cells in the central nervous system (CNS), which are involved in immune surveillance and maintenance of homeostasis in the CNS. Recently, a novel subpopulation of activated microglia named as disease-associated microglia (DAM), also known as activated response microglia (ARM) or microglial neurodegenerative phenotype (MGnD), was identified in AD model mice. These microglia closely associate with β-amyloid (Aβ) plaques and exhibit characteristic gene expression profiles accompanied with reduced expressions of homeostatic microglial genes. However, it remains unclear whether decreased homeostatic microglia functions or increased DAM/ARM/MGnD functions correlate with the degree of neuronal loss in AD. To translate the results of rodent studies to human AD, precuneus, the brain region vulnerable to β-amyloid accumulation in preclinical AD, is of high interest, as it can provide novel insights into the mechanisms of microglia response to Aβ in early AD. In this study, we performed comparative analyses of gene expression profiles of microglia among three representative neurodegenerative mouse models and the human precunei with early AD pathology. We proceeded to evaluate the identified genes as potential therapeutic targets for AD. We believe that our findings will provide important resources to better understand the role of glial dysfunction in AD.

Organelle contact sites are multifunctional platforms for maintaining cellular homeostasis. Alternations of the mitochondria-associated membranes (MAM), one of the organelle contact sites where the endoplasmic reticulum (ER) is tethered to the mitochondria, have been involved in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the detailed mechanisms through which MAM integrity is disrupted in ALS have not been fully elucidated. Here, we examined whether AAA ATPase domain-containing protein 3A (ATAD3A), a mitochondrial membrane AAA ATPase accumulating at the MAM, is involved in ALS. We found that sigma-1 receptor (σ1R), an ER-resident MAM protein causative for inherited juvenile ALS, required ATAD3A to maintain the MAM. In addition, σ1R retained ATAD3A as a monomer, which is associated with an inhibition of mitochondrial fragmentation. ATAD3A dimerization and mitochondrial fragmentation were significantly induced in σ1R-deficient or SOD1-linked ALS mouse spinal cords. Overall, these observations indicate that MAM induction by σ1R depends on ATAD3A and that σ1R maintains ATAD3A as a monomer to inhibit mitochondrial fragmentation. Our findings suggest that targeting σ1R-ATAD3A axis would be promising for a novel therapeutic strategy to treat mitochondrial dysfunction in neurological disorders, including ALS.

Current antipsychotics used to treat schizophrenia have associated problems, including serious side effects and treatment resistance. We recently identified a significant association of schizophrenia with exonic copy number variations in the Rho GTPase activating protein 10 (ARHGAP10) gene using genome-wide analysis. ARHGAP10 encodes a RhoGAP superfamily member that is involved in small GTPase signaling. In mice, Arhgap10 gene variations result in RhoA/Rho-kinase pathway activation. We evaluated the pharmacokinetics of fasudil and hydroxyfasudil using liquid chromatography-tandem mass spectrometry in mice. The antipsychotic effects of fasudil on hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits were also investigated in a MK-801-treated pharmacological mouse schizophrenia model. Fasudil and its major metabolite, hydroxyfasudil, were detected in the brain at concentrations above their respective Ki values for Rho-kinase after intraperitoneal injection of 10 mg kg-1 fasudil. Fasudil improved the hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits in MK-801-treated mice in a dose-dependent manner. Following oral administration of fasudil, brain hydroxyfasudil was detected at concentration above the Ki value for Rho-kinase whilst fasudil was undetectable. MK-801-induced hyperlocomotion was also improved by oral fasudil administration. These results suggest that fasudil has antipsychotic-like effects on the MK-801-treated pharmacological mouse schizophrenia model. There are two isoforms in Rho-kinase, and further investigation is needed to clarify the isoforms involved in the antipsychotic-like effects of fasudil in the MK-801-treated mouse schizophrenia model.

The mitochondria-associated membrane (MAM) is a functional subdomain of the endoplasmic reticulum membrane that tethers to the mitochondrial outer membrane and is essential for cellular homeostasis. A defect in MAM is involved in various neurological diseases, including amyotrophic lateral sclerosis (ALS). Recently, we and others reported that MAM was disrupted in the models expressing several ALS-linked genes, including SOD1, SIGMAR1, VAPB, TARDBP, and FUS, suggesting that MAM disruption is deeply involved in the pathomechanism of ALS. However, it is still uncertain whether MAM disruption is a common pathology in ALS, mainly due to the absence of a simple, quantitative tool for monitoring the status of MAM. In this study, to examine the effects of various ALS-causative genes on MAM, we created the following two novel MAM reporters: MAMtracker-Luc and MAMtracker-Green. The MAMtrackers could detect MAM disruption caused by suppression of SIGMAR1 or the overexpression of ALS-linked mutant SOD1 in living cells. Moreover, the MAMtrackers have an advantage in their ability to monitor reversible changes in the MAM status induced by nutritional conditions. We used the MAMtrackers with an expression plasmid library of ALS-causative genes and noted that 76% (16/21) of the genes altered MAM integrity. Our results suggest that MAM disruption is a common pathological feature in ALS. Furthermore, we anticipate our MAMtrackers, which are suitable for high-throughput assays, to be valuable tools to understand MAM dynamics.

We recently found a significant association between exonic copy-number variations in the Rho GTPase activating protein 10 (Arhgap10) gene and schizophrenia in Japanese patients. Special attention was paid to one patient carrying a missense variant (p.S490P) in exon 17, which overlapped with an exonic deletion in the other allele. Accordingly, we generated a mouse model (Arhgap10 S490P/NHEJ mice) carrying a missense variant and a coexisting frameshift mutation. We examined the spatiotemporal expression of Arhgap10 mRNA in the brain and found the highest expression levels in the cerebellum, striatum, and nucleus accumbens (NAc), followed by the frontal cortex in adolescent mice. The expression levels of phosphorylated myosin phosphatase-targeting subunit 1 and phosphorylated p21-activated kinases in the striatum and NAc were significantly increased in Arhgap10 S490P/NHEJ mice compared with wild-type littermates. Arhgap10 S490P/NHEJ mice exhibited a significant increase in neuronal complexity and spine density in the striatum and NAc. There was no difference in touchscreen-based visual discrimination learning between Arhgap10 S490P/NHEJ and wild-type mice, but a significant impairment of visual discrimination was evident in Arhgap10 S490P/NHEJ mice but not wild-type mice when they were treated with methamphetamine. The number of c-Fos-positive cells was significantly increased after methamphetamine treatment in the dorsomedial striatum and NAc core of Arhgap10 S490P/NHEJ mice. Taken together, these results suggested that schizophrenia-associated Arhgap10 gene mutations result in morphological abnormality of neurons in the striatum and NAc, which may be associated with vulnerability of cognition to methamphetamine treatment.

Microglia-mediated neuroinflammation has been implicated in the pathogenesis of Alzheimer's disease (AD). Although microglia in aging and neurodegenerative disease model mice show a loss of homeostatic phenotype and activation of disease-associated microglia (DAM), a correlation between those phenotypes and the degree of neuronal cell loss has not been clarified. In this study, we performed RNA sequencing of microglia isolated from three representative neurodegenerative mouse models, AppNL-G-F/NL-G-F with amyloid pathology, rTg4510 with tauopathy, and SOD1G93A with motor neuron disease by magnetic activated cell sorting. In parallel, gene expression patterns of the human precuneus with early Alzheimer's change (n = 11) and control brain (n = 14) were also analyzed by RNA sequencing. We found that a substantial reduction of homeostatic microglial genes in rTg4510 and SOD1G93A microglia, whereas DAM genes were uniformly upregulated in all mouse models. The reduction of homeostatic microglial genes was correlated with the degree of neuronal cell loss. In human precuneus with early AD pathology, reduced expression of genes related to microglia- and oligodendrocyte-specific markers was observed, although the expression of DAM genes was not upregulated. Our results implicate a loss of homeostatic microglial function in the progression of AD and other neurodegenerative diseases. Moreover, analyses of human precuneus also suggest loss of microglia and oligodendrocyte functions induced by early amyloid pathology in human.

Reelin, an extracellular matrix protein, is secreted by Cajal-Retzius cells and plays crucial roles in the development of brain structures and neuronal functions. Reductions in Reelin cause the brain dysfunctions associated with mental disorders, such as schizophrenia. A recent genome-wide copy number variation analysis of Japanese schizophrenia patients identified a novel deletion in RELN encoding Reelin. To clarify the pathophysiological role of the RELN deletion, we developed transgenic mice carrying the RELN deletion (Reln-del) and found abnormalities in their brain structures and social behavior. In the present study, we performed an in vitro analysis of Reelin expression, intracellular Reelin signaling, and the morphology of primary cultured cortical neurons from wild-type (WT) and Reln-del mice. Reelin protein levels were lower in Reln-del neurons than in WT neurons. Dab1 expression levels were significantly higher in Reln-del neurons than in WT neurons, suggesting that Reelin signaling was decreased in Reln-del neurons. Reelin was mainly expressed in γ-aminobutyric acid (GABA)-ergic inhibitory neurons, but not in parvalbumin (PV)-positive neurons. A small proportion of Ca2+/calmodulin-dependent protein kinase II α subunit (CaMKIIα)-positive excitatory neurons also expressed Reelin. In comparisons with WT neurons, significant decreases were observed in neurite lengths and branch points as well as in the number of postsynaptic density protein 95 (PSD95) immunoreactive puncta in Reln-del neurons. A disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3) is a protease that inactivates Reelin by cleavage at the N-t site. The knockdown of ADAMTS-3 by short hairpin RNAs suppressed Reelin cleavage in conditioned medium and reduced Dab1 expression, indicating that Reelin signaling was enhanced in the primary cultured cortical neurons of WT and heterozygous Reln-del. Accordingly, the inhibition of ADAMTS-3 may be a potential candidate in the clinical treatment of schizophrenia by enhancing Reelin signaling in the brain.

BACKGROUND: Immune molecules, such as cytokines, complement, and major histocompatibility complex (MHC) proteins, in the central nervous system are often associated with neuropsychiatric disorders. Neuronal MHC class I (MHCI), such as H-2D, regulate neurite outgrowth, the establishment and function of cortical connections, and activity-dependent refinement in mice. We previously established mice expressing MHCI specifically in astrocytes of the media prefrontal cortex (mPFC) using the adeno-associated virus (AAV) vector under the control of the GfaABC1D promoter. Mice expressing the soluble form of H-2D (sH-2D) in the mPFC (sH-2D-expressing mice) showed abnormal behaviors, including social interaction deficits and cognitive dysfunctions. However, the pathophysiological significance of astroglial MHCI on higher brain functions, such as learning, memory, and behavioral flexibility, remains unclear. Therefore, cognitive function in mice expressing sH-2D in astrocytes of the mPFC was tested using the visual discrimination (VD) task. METHODS: sH-2D-expressing mice were subjected to the VD and reversal learning tasks, and morphological analysis. RESULTS: In the pretraining, sH-2D-expressing mice required significantly more trials to reach the learning criterion than control mice. The total number of sessions, trials, normal trials, and correction trials to reach the VD criterion were also significantly higher in sH-2D-expressing mice than in control mice. A morphological study showed that dendritic complexity and spine density were significantly reduced in the dorsal striatum of sH-2D-expressing mice. CONCLUSION: Collectively, the present results suggest that the overexpression of astroglial MHCI in the mPFC results in impaired VD learning, which may be accompanied by decreased dendritic complexity in the dorsal striatum and mPFC.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline with accumulation of amyloid beta (Aβ) and neurofibrillary tangles that usually begins 15-30 years before clinical diagnosis. Rodent models that recapitulate aggressive Aβ and/or the pathology of neurofibrillary tangles are essential for AD research. Accordingly, non-invasive early detection systems in these animal models are required to evaluate the phenotypic changes, elucidate the mechanism of disease progression, and facilitate development of novel therapeutic approaches. Although many behavioral tests efficiently reveal cognitive impairments at the later stage of the disease in AD models, it has been challenging to detect such impairments at the early stage. To address this issue, we subjected 4-6-month-old male AppNL-G-F/NL-G-F knock-in (App-KI) mice to touchscreen-based location discrimination (LD), different object-location paired-associate learning (dPAL), and reversal learning tests, and compared the results with those of the classical Morris water maze test. These tests are mainly dependent on the brain regions prone to Aβ accumulation at the earliest stages of the disease. At 4-6 months, considered to represent the early stage of disease when mice exhibit initial deposition of Aβ and slight gliosis, the classical Morris water maze test revealed no difference between groups, whereas touchscreen-based LD and dPAL tasks revealed significant impairments in task performance. Our report is the first to confirm that a systematic touchscreen-based behavioral test battery can sensitively detect the early stage of cognitive decline in an AD-linked App-KI mouse model. This system could be applied in future translational research.

Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and a subtype of frontotemporal lobar degeneration (FTLD). Recent studies have suggested that the formation of cytoplasmic TDP-43 aggregates is dependent on a liquid-liquid phase separation (LLPS) mechanism. However, it is unclear whether TDP-43 pathology is induced through a single intracellular mechanism such as LLPS. To identify intracellular mechanisms responsible for TDP-43 aggregation, we established a TDP-43 aggregation screening system using a cultured neuronal cell line stably expressing EGFP-fused TDP-43 and a mammalian expression library of the inherited ALS/FTLD causative genes, and performed a screening. We found that microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) co-aggregated with TDP-43. MRPs and RBPs sequestered TDP-43 into the cytoplasmic aggregates through distinct mechanisms, such as microtubules and LLPS, respectively. The MRPs-induced TDP-43 aggregates were co-localized with aggresomal markers and dependent on histone deacetylase 6 (HDAC6), suggesting that aggresome formation induced the co-aggregation. However, the MRPs-induced aggregates were not affected by 1,6-hexanediol, an LLPS inhibitor. On the other hand, the RBPs-induced TDP-43 aggregates were sensitive to 1,6-hexanediol, but not dependent on microtubules or HDAC6. In sporadic ALS patients, approximately half of skein-like TDP-43 inclusions were co-localized with HDAC6, but round and granular type inclusion were not. Moreover, HDAC6-positive and HDAC6-negative inclusions were found in the same ALS patient, suggesting that the two distinct pathways are both involved in TDP-43 pathology. Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS) are involved in inducing the TDP-43 pathologies.

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.

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.

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43), a DNA/RNA binding protein, is a pathological signature of amyotrophic lateral sclerosis (ALS). Missense mutations in the TARDBP gene are also found in inherited and sporadic ALS, indicating that dysfunction in TDP-43 is causative for ALS. To model TDP-43-linked ALS in rodents, we generated TDP-43 knock-in mice with inherited ALS patient-derived TDP-43M337V mutation. Homozygous TDP-43M337V mice developed normally without exhibiting detectable motor dysfunction and neurodegeneration. However, splicing of mRNAs regulated by TDP-43 was deregulated in the spinal cords of TDP-43M337V mice. Together with the recently reported TDP-43 knock-in mice with ALS-linked mutations, our finding indicates that ALS patient-derived mutations in the TARDBP gene at a carboxyl-terminal domain of TDP-43 may cause a gain of splicing function by TDP-43, however, were insufficient to induce robust neurodegeneration in mice.

<p>Schizophrenia is a severe mental illness that affects about 1% of the population. Genetic and environmental factors contribute to the development of schizophrenia. However, the exact pathoetiology remains unclear. We generated Rho GTPase-activating protein 10 (ARHGAP10) mutant mice carrying similar variations found in Japanese schizophrenia patients. In the present study, we examined spatiotemporal expression of ARHGAP10 mRNA in the brain of mice. The expression levels of ARHGAP10 mRNA were higher in the striatum (ST) and nucleus accumbens (NAc) than those in other brain regions. We performed a series of behavior test to evaluate cognitive and emotional function in ARHGAP10 mutant mice. They showed an increase in anxiety level, and manifested potentiation of methamphetamine-induced hyperlocomotion and visual discrimination task. Morphological analysis revealed that methamphetamine-treated ARHGAP10 mutant mice showed an increase in the number of c-Fos-positive-cells in the dorsal medial striatum (dmST) and NAc core than those in wild-type littermates. Golgi staining indicated that ARHGAP10 mutant mice showed an increase in neuronal complexity and spine density in the same brain regions compared to the wild-type mice. These results suggest that ARHGAP10 gene variations may lead to the development of cognitive and emotional deficits with morphological abnormality in the dmST and NAc core neurons.</p>

<p>Alzheimer's disease (AD) is a progressive disease characterized by loss of memory and other important mental functions. Accumulation of amyloid beta (Aβ) and fibrillary tangles are the main pathological hallmarks of this disease. Emerging data suggest that the disease process begins years before clinical diagnostic confirmation; thus, methods to improve early detection would provide opportunities for early intervention to delay progressive cognitive decline and disease onset. In our research we assessed the cognitive abilities of APP^{NL-G-F/NL-G-F} knock-in (APP-KI) mice with a touch screen-based automated test battery and water maze test. These tests are mainly dependent on the brain regions that are prone to Aβ accumulation at the earliest stages of the disease. We subjected male 6- and 11-months old APP-KI mice in water maze test where only older mice showed significantly worsened behavior than wild-type mice. However, using touchscreen based behavioral test it was possible to detect cognitive impairment in APP-KI mice at an early (4 months) stage while classical behavioral tests shows comparable results between wild-type and disease model mice. </p>

Reelin protein (RELN), an extracellular matrix protein, plays multiple roles that range from embryonic neuronal migration to spine formation in the adult brain. Results from genetic studies have suggested that RELN is associated with the risk of psychiatric disorders, including schizophrenia (SCZ). We previously identified a novel exonic deletion of RELN in a patient with SCZ. High-resolution copy number variation analysis revealed that this deletion included exons 52 to 58, which truncated the RELN in a similar manner to the Reln Orleans mutation (Relnrl-Orl). We examined the clinical features of this patient and confirmed a decreased serum level of RELN. To elucidate the pathophysiological role of the exonic deletion of RELN in SCZ, we conducted behavioral and neurochemical analyses using heterozygous Relnrl-Orl/+ mice. These mice exhibited abnormalities in anxiety, social behavior, and motor learning; the deficits in motor learning were ameliorated by antipsychotics. Methamphetamine-induced hyperactivity and dopamine release were significantly reduced in the Relnrl-Orl/+ mice. In addition, the levels of GABAergic markers were decreased in the brain of these mice. Taken together, our results suggest that the exonic deletion of RELN plays a pathological role, implicating functional changes in the dopaminergic and GABAergic systems, in the pathophysiology of SCZ.

In the central nervous system, major histocompatibility complex class I (MHCI) molecules are mainly expressed in neurons, and neuronal MHCI have roles in synapse elimination and plasticity. However, the pathophysiological significance of astroglial MHCI remains unclear. We herein demonstrate that MHCI expression is up-regulated in astrocytes in the medial prefrontal cortex (mPFC) following systemic immune activation by an intraperitoneal injection of polyinosinic-polycytidylic acid (polyI:C) or hydrodynamic interferon (IFN)-γ gene delivery in male C57/BL6J mice. In cultured astrocytes, MHCI/H-2D largely co-localized with exosomes. To investigate the role of astroglial MHCI, H-2D, or sH-2D was expressed in the mPFC of male C57/BL6J mice using an adeno-associated virus vector under the control of a glial fibrillary acidic protein promoter. The expression of astroglial MHCI in the mPFC impaired sociability and recognition memory in mice. Regarding neuropathological changes, MHCI expression in astrocytes significantly activated microglial cells, decreased parvalbumin-positive cell numbers, and reduced dendritic spine density in the mPFC. A treatment with GW4869 that impairs exosome synthesis ameliorated these behavioral and neuropathological changes. These results suggest that the overexpression of MHCI in astrocytes affects microglial proliferation as well as neuronal numbers and spine densities, thereby leading to social and cognitive deficits in mice, possibly via exosomes created by astrocytes.