村松 慎一

OBJECTIVE: The purpose of this study was to extract important patient questionnaire items by creating random forest models for predicting pattern diagnosis considering an interaction between deficiency-excess and cold-heat patterns. DESIGN: A multi-centre prospective observational study. SETTING: Participants visiting six Kampo speciality clinics in Japan from 2012 to 2015. MAIN OUTCOME MEASURE: Deficiency-excess pattern diagnosis made by board-certified Kampo experts. METHODS: We used 153 items as independent variables including, age, sex, body mass index, systolic and diastolic blood pressures, and 148 subjective symptoms recorded through a questionnaire. We sampled training data with an equal number of the different patterns from a 2 × 2 factorial combination of deficiency-excess and cold-heat patterns. We constructed the prediction models of deficiency-excess and cold-heat patterns using the random forest algorithm, extracted the top 10 essential items, and calculated the discriminant ratio using this prediction model. RESULTS: BMI and blood pressure, and subjective symptoms of cold or heat sensations were the most important items in the prediction models of deficiency-excess pattern and of cold-heat patterns, respectively. The discriminant ratio was not inferior compared with the result ignoring the interaction between the diagnoses. CONCLUSIONS: We revised deficiency-excess and cold-heat pattern prediction models, based on balanced training sample data obtained from six Kampo speciality clinics in Japan. The revised important items for diagnosing a deficiency-excess pattern and cold-heat pattern were compatible with the definition in the 11th version of international classification of diseases.

The timing and characteristics of neuronal death in Alzheimer's disease (AD) remain largely unknown. Here we examine AD mouse models with an original marker, myristoylated alanine-rich C-kinase substrate phosphorylated at serine 46 (pSer46-MARCKS), and reveal an increase of neuronal necrosis during pre-symptomatic phase and a subsequent decrease during symptomatic phase. Postmortem brains of mild cognitive impairment (MCI) rather than symptomatic AD patients reveal a remarkable increase of necrosis. In vivo imaging reveals instability of endoplasmic reticulum (ER) in mouse AD models and genome-edited human AD iPS cell-derived neurons. The level of nuclear Yes-associated protein (YAP) is remarkably decreased in such neurons under AD pathology due to the sequestration into cytoplasmic amyloid beta (Aβ) aggregates, supporting the feature of YAP-dependent necrosis. Suppression of early-stage neuronal death by AAV-YAPdeltaC reduces the later-stage extracellular Aβ burden and cognitive impairment, suggesting that preclinical/prodromal YAP-dependent neuronal necrosis represents a target for AD therapeutics.

Clustered protocadherins (Pcdhs), a large group of adhesion molecules, are important for axonal projections and dendritic spread, but little is known about how they influence neuronal activity. The Pcdhβ cluster is strongly expressed in the hippocampus, and in vivo Ca2+ imaging in Pcdhβ-deficient mice revealed altered activity of neuronal ensembles but not of individual cells in this region in freely moving animals. Specifically, Pcdhβ deficiency increased the number of large-size neuronal ensembles and the proportion of cells shared between ensembles. Furthermore, Pcdhβ-deficient mice exhibited reduced repetitive neuronal population activity during exploration of a novel context and were less able to discriminate contexts in a contextual fear conditioning paradigm. These results suggest that one function of Pcdhβs is to modulate neural ensemble activity in the hippocampus to promote context discrimination.

Targeting cytoplasmic protein-protein interactions with antibodies remains technically challenging, since antibodies expressed in the cytosol frequently form insoluble aggregates. Existing engineering methods are based on the notion that the estimated net charge at pH 7.4 affects stability; as such, they are unable to overcome this problem. Herein, we report a versatile method for engineering an ultra-stable cytoplasmic antibody (STAND), with a strong estimated net negative charge at pH 6.6, by fusing peptide tags with a highly negative charge and a low isoelectric point. Without the need for complicated amino acid substitutions, we convert aggregation-prone antibodies to STANDs that are useful for inhibiting in vivo transmitter release, modulating animal behaviour, and inhibiting in vivo cancer proliferation driven by mutated Kras-long recognised as an "undruggable" oncogenic protein. The STAND method shows promise for targeting endogenous cytoplasmic proteins in basic biology and for developing future disease treatments.

The number of patients with depressive disorders is increasing. However, the mechanism of depression onsets has not been completely revealed. We previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. In this study, we revealed the involvement of Shati/Nat8l in the vulnerability to major depression. Shati/Nat8l mRNA was increased only in the striatum of mice, which were exposed to chronic social defeat stress. Shati/Nat8l-overexpressed mice showed impairment in social interaction and sucrose preference after the subthreshold social defeat (microdefeat) stress. These depression-like behaviors were restored by fluvoxamine and LY341495 injection prior to these tests. Furthermore, the intracerebral administration of only fluvoxamine, but not of LY341495, to the dorsal striatum and direct infusion of LY341495 to the dorsal raphe also rescued. Taken together, Shati/Nat8l in the striatum has an important role in the vulnerability to depression onsets by regulating the origin of serotonergic neuronal system via GABAergic projection neuron in the dorsal raphe from the dorsal striatum.

AIM: We previously reported that methamphetamine (METH)-induced conditioned place preference was attenuated by Shati/Nat8l overexpression in the medial prefrontal cortex (mPFC). Shati/Nat8l overexpression in the mPFC expressed lower levels of both glutamate and dopamine (DA) in the nucleus accumbens (NAc) and attenuated METH-induced DA elevation. We suggested a mechanism in which a decline of glutamate levels in the NAc decreases extracellular DA levels. However, the hypothesis has not confirmed. METHODS: We conducted a recovery experiments by pre-microinjection of an mGluR group II antagonist, LY341495, into the NAc shell of mPFC-Shati/Nat8l-overexpressed mice followed by METH injection and DA levels measurement by in vivo microdialysis. RESULTS: Pretreatment with LY341495 was able to restore METH-induced DA increase. Furthermore, mice injected with an adeno-associated virus vector containing GFP (AAV-GFP vector) in the mPFC expressed a colocalization of GFP with DARPP-32 a medium spiny neuron (MSN) marker. Next, co-immunostaining of DARPP-32 and neuronal nitric oxide synthase (nNOS: expressed in a subtype of gamma-Aminobutyric acid (GABA interneurons) in ventral tegmental area (VTA) showed a colocalization of nNOS and DARPP-32. CONCLUSION: These results provided a proof that Shati/Nat8l attenuation of METH-induced DA increase is mediated by mGluR group II in the NAc. Moreover, immunohistochemical study showed a direct connection of mPFC projection neurons with NAc MSN and a connection of MSN projection neurons with a subtype of GABA interneurons in VTA.

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4+/-/ARF5-/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4+/-/ARF5-/- mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4+/-/ARF5-/- mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na+ channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4+/-/ARF5-/- mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4+/-/ARF5-/- mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS.SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4+/-/ARF5-/- mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4+/-/ARF5-/- mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4+/-/ARF5-/- mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.

OBJECTIVE: The purpose of the present study was to compare important patient questionnaire items by creating a random forest model for predicting deficiency-excess pattern diagnosis in six Kampo specialty clinics. DESIGN: A multi-centre prospective observational study. SETTING: Participants who visited six Kampo specialty clinics in Japan from 2012 to 2015. MAIN OUTCOME MEASURE: Deficiency-excess pattern diagnosis made by board-certified Kampo experts. METHODS: To predict the deficiency-excess pattern diagnosis by Kampo experts, we used 153 items as independent variables, namely, age, sex, body mass index, systolic and diastolic blood pressures, and 148 subjective symptoms recorded through a questionnaire. We extracted the 30 most important items in each clinic's random forest model and selected items that were common among the clinics. We integrated participating clinics' data to construct a prediction model in the same manner. We calculated the discriminant ratio using this prediction model for the total six clinics' data and each clinic's independent data. RESULTS: Fifteen items were commonly listed in top 30 items in each random forest model. The discriminant ratio of the total six clinics' data was 82.3%; moreover, with the exception of one clinic, the independent discriminant ratio of each clinic was approximately 80% each. CONCLUSIONS: We identified common important items in diagnosing a deficiency-excess pattern among six Japanese Kampo clinics. We constructed the integrated prediction model of deficiency-excess pattern.

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured β cell line (MIN6) and β cell-specific Tardbp knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage-dependent calcium channels and thus plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.

To understand the molecular processes that link Aβ amyloidosis, tauopathy and neurodegeneration, we screened for tau-interacting proteins by immunoprecipitation/LC-MS. We identified the carboxy-terminal PDZ ligand of nNOS (CAPON) as a novel tau-binding protein. CAPON is an adaptor protein of neuronal nitric oxide synthase (nNOS), and activated by the N-methyl-D-aspartate receptor. We observed accumulation of CAPON in the hippocampal pyramidal cell layer in the AppNL-G-F -knock-in (KI) brain. To investigate the effect of CAPON accumulation on Alzheimer's disease (AD) pathogenesis, CAPON was overexpressed in the brain of AppNL-G-F mice crossbred with MAPT (human tau)-KI mice. This produced significant hippocampal atrophy and caspase3-dependent neuronal cell death in the CAPON-expressing hippocampus, suggesting that CAPON accumulation increases neurodegeneration. CAPON expression also induced significantly higher levels of phosphorylated, oligomerized and insoluble tau. In contrast, CAPON deficiency ameliorated the AD-related pathological phenotypes in tauopathy model. These findings suggest that CAPON could be a druggable AD target.

Parkinson's disease (PD) is the second common neurodegenerative disorder. Deficit of the nigro-striatal dopaminergic neurons causes the motor symptoms of PD. While the oxidative stress is thought to be deeply involved in the etiology of PD, molecular targets for the oxidative insults has not been fully elucidated. 6R-5,6,7,8-Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase (TH), the rate-limiting enzyme for production of dopamine, and easily oxidized to its dihydro-form. In this study, we examined the alteration in the metabolism of BH4 caused by a parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP reduced the dopamine content and the in vivo activity of TH in the striatum prior to degeneration of the dopaminergic neurons. We found that administration of BH4 could restore the dopamine content and in vivo TH activity in the striatum of MPTP-treated mice. Unexpectedly, when BH4 was administered with MPTP, BH4 contents in the brain were far higher than those injected without MPTP even at 23 h after the last injection. Because MPTP has been shown to increase ROS production in the dopaminergic neurons, we assumed that the increased ROS oxidizes BH4 into its dihydro-form, excreted from the dopaminergic neurons, taken-up by the neighboring cells, reduced back to BH4, and then accumulated in the brain. We also investigated the action of MPTP in mice lacking quinonoid-dihydropteridine reductase (Qdpr), an enzyme catalyzing regeneration of BH4 from quinonoid dihydrobiopterin. The dopamine depletion induced by MPTP was severer in Qdpr-deficient mice than in wild-type mice. The present data suggest that perturbation of the BH4 metabolism would be the cause of early and persistent dopamine depletion in the striatum.

In patients with aromatic l-amino acid decarboxylase (AADC) deficiency, a decrease in catecholamines and serotonin levels in the brain leads to developmental delay and movement disorders. The beneficial effects of gene therapy in patients from 1 to 8 years of age with homogeneous severity of disease have been reported from Taiwan. We conducted an open-label phase 1/2 study of population including adolescent patients with different degrees of severity. Six patients were enrolled: four males (ages 4, 10, 15 and 19 years) and one female (age 12 years) with a severe phenotype who were not capable of voluntary movement or speech, and one female (age 5 years) with a moderate phenotype who could walk with support. The patients received a total of 2 × 1011 vector genomes of adeno-associated virus vector harbouring DDC via bilateral intraputaminal infusions. At up to 2 years after gene therapy, the motor function was remarkably improved in all patients. Three patients with the severe phenotype were able to stand with support, and one patient could walk with a walker, while the patient with the moderate phenotype could run and ride a bicycle. This moderate-phenotype patient also showed improvement in her mental function, being able to converse fluently and perform simple arithmetic. Dystonia disappeared and oculogyric crisis was markedly decreased in all patients. The patients exhibited transient choreic dyskinesia for a couple of months, but no adverse events caused by vector were observed. PET with 6-[18F]fluoro-l-m-tyrosine, a specific tracer for AADC, showed a persistently increased uptake in the broad areas of the putamen. In our study, older patients (>8 years of age) also showed improvement, although treatment was more effective in younger patients. The genetic background of our patients was heterogeneous, and some patients suspected of having remnant enzyme activity showed better improvement than the Taiwanese patients. In addition to the alleviation of motor symptoms, the cognitive and verbal functions were improved in a patient with the moderate phenotype. The restoration of dopamine synthesis in the putamen via gene transfer provides transformative medical benefit across all patient ages, genotypes, and disease severities included in this study, with the most pronounced improvements noted in moderate patients.10.1093/brain/awy331_video1awy331media15991361892001.

Previous gain-of-function studies using an optogenetic technique showed that manipulation of the hippocampal dentate gyrus or CA1 cell ensembles is important for memory reactivation and to generate synthetic or false memory. However, gain-of-function study manipulating CA3 cell ensembles has not been reported. The CA3 area of the hippocampus comprises a recurrent excitatory circuit, which is thought to be important for the generation of associations among the stored information within one brain region. We investigated whether the coincident firing of cell ensembles in one brain region, hippocampal CA3, associates distinct events. CA3 cell ensembles responding to context exploration and during contextual fear conditioning were labeled with channelrhodopsin-2 (ChR2)-mCherry. The synchronous activation of these ensembles induced freezing behavior in mice in a neutral context, in which a foot shock had never been delivered. The recall of this artificial associative fear memory was context specific. In vivo electrophysiological recordings showed that 20-Hz optical stimulation of ChR2-mCherry-expressing CA3 neurons, which is the same stimulation protocol used in behavioral experiment, induced long-term potentiation at CA3-CA3 synapses. Altogether, these results demonstrate that the synchronous activation of ensembles in one brain region, CA3 of the hippocampus, is sufficient for the association of distinct events. The results of our electrophysiology potentially suggest that this artificial association of memory events might be induced by the strengthening of synaptic efficacy between CA3 ensembles via recurrent circuit.

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

Aromatic L-acid decarboxylase (AADC) deficiency causes severe motor disturbances in affected children. A putamen-targeted gene therapy improves the motor function of patients. The present study investigated the electrical properties of dopaminergic (DA) neurons in the substantia nigra compacta (SNc) of mice with an AADC deficiency (DdcKI). The basal firing of DA neurons, which determines DA release in the putamen, was abnormal in the DdcKI mice, including a low frequency and irregular firing pattern, because of a decrease in the after-hyperpolarization (AHP) amplitude of action potentials (APs). The frequency of spontaneous excitatory postsynaptic currents (sEPSCs) increased and that of spontaneous inhibitory PSCs (sIPSCs) decreased in the SNc DA neurons from the DdcKI mice, suggesting an elevation in glutamatergic excitatory stimuli and a reduction in GABAergic inhibitory stimuli, respectively. Altered expression patterns of genes encoding receptors and channels were also observed in the DdcKI mice. Administration of a widespread neuron-specific gene therapy to the brains of the DdcKI mice partially corrected these electric abnormalities. The overexcitability of SNc DA neurons in the presence of generalized dopamine deficiency likely underlies the occurrence of motor disturbances.

Early-phase pathologies of Alzheimer's disease (AD) are attracting much attention after clinical trials of drugs designed to remove beta-amyloid (Aβ) aggregates failed to recover memory and cognitive function in symptomatic AD patients. Here, we show that phosphorylation of serine/arginine repetitive matrix 2 (SRRM2) at Ser1068, which is observed in the brains of early phase AD mouse models and postmortem end-stage AD patients, prevents its nuclear translocation by inhibiting interaction with T-complex protein subunit α. SRRM2 deficiency in neurons destabilized polyglutamine binding protein 1 (PQBP1), a causative gene for intellectual disability (ID), greatly affecting the splicing patterns of synapse-related genes, as demonstrated in a newly generated PQBP1-conditional knockout model. PQBP1 and SRRM2 were downregulated in cortical neurons of human AD patients and mouse AD models, and the AAV-PQBP1 vector recovered RNA splicing, the synapse phenotype, and the cognitive decline in the two mouse models. Finally, the kinases responsible for the phosphorylation of SRRM2 at Ser1068 were identified as ERK1/2 (MAPK3/1). These results collectively reveal a new aspect of AD pathology in which a phosphorylation signal affecting RNA splicing and synapse integrity precedes the formation of extracellular Aβ aggregates and may progress in parallel with tau phosphorylation.

BACKGROUND: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene, encoding glucose transporter type 1 (GLUT1), was expressed under the human endogenous GLUT1 promoter (AAV-GLUT1). We examined whether AAV-GLUT1 administration could lead to functional improvement in GLUT1-deficient mice. METHODS: We extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine-mutant AAV9/3 vector in which human SLC2A1-myc-DDK was expressed under the human GLUT1 promoter (AAV-GLUT1). AAV-GLUT1 was administered to GLUT1-deficient mice (GLUT1+/- mice) via intracerebroventricular injection (1.85 × 1010 vg/mouse or 6.5 × 1010 vg/mouse). We analyzed exogenous GLUT1 mRNA and protein expression in the brain and other major organs. We also examined improvements of cerebral microvasculature, motor function using rota-rod and footprint tests, as well as blood and cerebrospinal fluid (CSF) glucose levels. Additionally, we confirmed exogenous GLUT1 protein distribution in the brain and other organs after intracardiac injection (7.8 × 1011 vg/mouse). RESULTS: Exogenous GLUT1 protein was strongly expressed in the cerebral cortex, hippocampus and thalamus. It was mainly expressed in endothelial cells, and partially expressed in neural cells and oligodendrocytes. Motor function and CSF glucose levels were significantly improved following intracerebroventricular injection. Exogenous GLUT1 expression was not detected in other organs after intracerebroventricular injection of AAV-GLUT1, whereas it was detected in the liver and muscle tissue after intracardiac injection. CONCLUSIONS: Exogenous GLUT1 expression after AAV-GLUT1 injection approximated that of physiological human GLUT1 expression. Local central nervous system administration of AAV-GLUT1 improved CSF glucose levels and motor function of GLUT1-deficient mice and minimized off-target effects.

Shati/Nat8l was originally isolated as a methamphetamine-related-molecule from the nucleus accumbens of mice. Since then, Shati/Nat8l has been characterized as an N-acetyltransferase-8-like protein (Nat8l) that catalyzes N-acetylaspartate (NAA) synthesis from aspartate and acetyl-coenzyme A. It has been shown that elevated NAA levels detected by proton magnetic resonance spectroscopy (1H-MRS) brain imaging indicates increased neuronal activity. Our group produced Shati/Nat8l knock out mice (Shati/Nat8l KO mice), which exhibit hyper locomotion, anxiety behaviors, and social dysfunction. These mice have a high sensitivity to methamphetamine, as evidenced by their results in assessments of locomotor activity and conditioned place preference, as well as their elevated dopamine levels. We used an adeno-associated virus (AAV) vector containing Shati/Nat8l (AAV-Shati/Nat8l) to overexpress the protein in different brain regions such as the striatum and the nucleus accumbens, in order to investigate their involvement in methamphetamine-induced behavioral and pharmacological changes. We showed that overexpression of accumbal Shati/Nat8l attenuates methamphetamine-induced behaviors. Recent clinical studies have revealed further novel roles of Shati/Nat8l in psychiatric and neuronal diseases. We are just beginning to appreciate the various actions of this intriguing, recently discovered molecule in the central nervous system.