Lewy body disease including Parkinson's disease (PD) and dementia with Lewy body (DLB) share not only imaging biomarkers including MIBG myocardial scintigraphy and dopamine transporter scintigraphy (DaT SPECT), but prodromal signs such as constipation, REM sleep behavior disorder (RBD), and hyposmia, which precede the onset of motor or cognitive symptoms by as early as 20 years. It is now widely known that there is diversity in the mode of progression in the prodromal phase of Lewy body disease. To gain insight in such diversity, we created a high-risk cohort of Lewy body disease by conducting a questionnaire survey of annual health checkup examinees without neurological symptoms using self-reported questionnaires on prodromal symptoms (NaT-PROBE study). Subjects ≥50 years of age with ≥2 prodromal symptoms (dysautonomia, hyposmia, and RBD), were classified as high-risk. Among 11,452 subjects ≥50 years of age, 759 (6.6%) were classified as high-risk. These subjects had worse values of BDI-II and ESS, in addition to SCOPA-AUT, SAOQ, and RBDSQ. At the second stage of investigation using imaging and fluid biomarkers, about one-third of high-risk subjects showed deficit in MIBG and/or DaT SPECT. In plasma biomarker analysis, AD-related biomarker levels were not elevated in the high-risk group, but NfL levels were higher in the high-risk, PD, and DLB groups than in the low-risk group. NfL elevation was associated with MIBG abnormalities in the high-risk group. We also developed a novel questionnaire to screen mild motor symptoms in preclinical/prodromal subjects of Lewy body disease: Screening Questionnaire for Subtle Parkinsonism (SQSP). The score of SQSP was higher in the high-risk subjects than in the low-risk individuals who had no prodromal symptoms. It also showed association with DaT SPECT SBR values and MDS-UPDRS part 3 scores in the high-risk group. We are currently conducting a placebo-controlled randomized clinical trial of zonisamide for the high-risk subjects with imaging deficits as a preventive therapy (jRCTs041190126).

Fibroblasts, a type of stromal cells that exist in almost all tissues and organs, get activated and proliferate in response to tissue damage induced by many causes, including physical wounds, inflammation, and cancer invasion. The primary and fundamental role of activated fibroblasts is the repair of injured tissues in collaboration with other immune cells, such as macrophages. The problem is, however, the sustained activation of the fibroblasts, which leads to excessive deposition of extracellular matrix, fibrosis, and tissue stiffening that impair the physiological function of the affected organs. We have been recently focusing on the diverse roles of fibroblasts in various fibroinflammatory diseases and cancer and found that we have bad fibroblasts that promote disesae progression and good ones that inhibit it. Interestingly, a pharmacological approach to increase the number of good fibroblasts ameliorated stromal fibrosis and improved the sensitivity of intractable cancers such as pancreatic cancer to chemotherapy and immunotherapy in preclinical mouse models. Based on these findings, two clinical studies on patients with advanced pancreatic cancer have been initiated at Nagoya University and the University of Tokyo. This approach could also apply to treating other types of fibrotic diseases, such as cardiac and lung fibrosis. In this session, I will talk about recent progress on fibrosis research in our laboratory, which I hope will raise active discussion among the audience.

DNA-protein crosslinks (DPCs), formed when proteins become covalently bound to DNA, pose a significant threat to cellular function by blocking essential DNA transactions. Among the various DPC-inducing agents, endogenous aldehydes are of particular concern due to their continuous production by normal cellular metabolism. Recent clinical evidence has highlighted the importance of aldehyde detoxification pathways, as exemplified by AMeD syndrome, a newly identified disorder caused by mutations in both the ALDH2 and ADH5 genes. Patients with AMeD syndrome present with a complex phenotype including bone marrow failure, developmental delay and neurological abnormalities, suggesting a critical role for aldehyde metabolism in multiple organ systems. While previous research has established the importance of replication-coupled DPC repair pathways, our study reveals a novel mechanism for the removal of aldehyde-induced DPCs from actively transcribed regions by transcription-coupled repair (TCR). Using our newly developed DPC-seq method, we show that the conventional TCR pathway, together with VCP/p97 and the proteasome, efficiently removes DPCs from transcribed regions. Furthermore, we show that mice deficient in both aldehyde metabolism and TCR exhibit severe phenotypes reminiscent of human AMeD syndrome, particularly affecting haematopoietic stem cells. These findings demonstrate that both transcription-coupled DPC repair (TC-DPCR) and aldehyde clearance are critical for protection against metabolic genotoxins and thus explain the molecular pathogenesis of AMeD and other genetic disorders associated with defects in the TCR, such as Cockayne syndrome.

The cell, as the fundamental unit of life, is surrounded by a plasma membrane that functions as a barrier between the internal environment and the external surroundings. Plasma membrane is composed of a lipid bilayer and plays crucial roles in protecting the cell and regulating the movement of substances in and out of the cell.

The process of cell adhesion, a prerequisite for the formation of tissues, could be hindered by the surface static electrical charge resulting from the presence of charged molecules including phospholipids. Cell adhesion, therefore, requires many molecules that tether the opposing cell membranes to each other.

Consequently, cell-to-cell membrane fusion rarely occurs in typical living tissues, irrespective of the density of cell presence. However, it is also well-documented that cell fusion phenomena occur frequently during specific events, including fertilization, the development of osteoclasts and muscular cells. The process of membrane fusion, specifically that of the lipid bilayers, has been demonstrated to require "membrane fusion machinery," or "fusogens," which facilitates the sequential fusing of the lipid bilayers by anchoring paired membrane lipids in close proximity to the fusion initiation site. The composition of these membrane fusion molecules, however, remains to be elucidated.

The present study was thus aimed to understand the molecular basis of plasma membrane fusion. To this end, cultured myoblast cells were utilized to analyze the mechanism. Firstly, a semi-quantitative live assay system was established to monitor the cell-cell fusion event using HiBiT-LgBiT system (Promega Corp.). The assay (HiMy: HiBiT-dependent Myogenic cell fusion) system was then utilized to screen for molecules that modify cell fusion activity, employing a library of siRNAs and chemical compounds. Subsequently, the molecular characterization of two known 'fusogenic' membrane proteins, myomaker and myomixer, was performed with HiMy assay, confirming their fusogenic activity. The results from our current approach would be discussed.   

Cell-free protein synthesis (CFPS) is an innovative method in biotechnology, offering a more efficient alternative to traditional in vivo protein production. This approach involves synthesizing proteins in vitro by utilizing cellular machinery extracted from cells or purified components, bypassing the need for living organisms. Using CFPS, we have developed the "Ecobody technology," a novel system for swiftly acquiring monoclonal antibodies. Initially, B cells are sourced from animal or human blood or lymph nodes, and those binding to the target antigen are isolated using a cell sorter or magnetic beads. Subsequently, the fragment of antigen binding (fab) of the light and heavy chains of antibody in each cell are amplified using RT-PCR and PCR techniques. The amplified DNA fragments are then modified to incorporate the necessary DNA sequences for expression in E. coli CFPS. The fabs are synthesized in microwells via CFPS and subjected to multiple assays such as ELISA and immunostaining. Since the entire process can be easily automated by sequentially adding and mixing reagents, this technology has the potential to significantly enhance the throughput of monoclonal antibody acquisition. With the goal of implementing this technology into society, iBody Inc. (https://www.ibody.co.jp/) was established in 2018. Moreover, CFPS has been used form epitope determination of monoclonal antibodies and affinity improvement of Fab.

Upon epidermal formation in the skin, keratinocyte differentiates accompanying with the expressions of various functional proteins resulting in the stable barrier construction at the outermost surface of the body. Among the functional proteins in keratinocytes, transglutaminase (TG1) is the key enzyme that catalyzes the protein cross-linking of several structural proteins essential for barrier function (cornified envelop formation). Aberrant activity of this enzyme causes several skin diseases, which have been investigated to develop efficient therapy and drug. So far, we have studied properties of the catalytic reaction, substrate specificity, and functional significance during differentiation, using in vivoand in vitro model system. As in vitro system, we clarified the substrate preference of TG1, and established specific detection system of in situ activity and identification procedure of substrates. For in vivo system, because knock-out (KO) mice die after birth by insufficient barrier function, investigation using an inducible system for gene-disruption was required. We generated conditional KO mice, by Cre/loxP system, which is available for the analysis in adult. Phenotypes as inflammation and aberrant epithelium tissue were observed in addition to abnormal skin formation.      

Sarcopenia is an urgent medical and socioeconomic problem in rapidly aging societies. However, the underlying molecular mechanisms of sarcopenia remain unclear. A number of studies have shown that the progressive and systemic decrease in nicotinamide adenine dinucleotide (NAD⁺) levels and the resultant dysfunction of NAD⁺-consuming enzymes, such as sirtuins, is a driving force of age-associated pathophysiology. In this study, we demonstrated the functional connection between the hypothalamus and skeletal muscle through NAD⁺-related genes (Slc12a8 and Nampt), and its relationship to sarcopenia (Ito N et al., Cell Reports, 2022. Eguchi T et al, in revision). We genetically knocked down Slc12a8 or Nampt in the lateral hypothalamus (LH) and analyzed their effects on whole-body metabolism and skeletal muscle functions. Genetic knockdown of Slc12a8 or Nampt specifically in the LH caused a decrease in energy and carbohydrate expenditure. Furthermore, LH-specific Slc12a8- or Nampt-knockdown mice showed significant reductions in skeletal muscle functions, including muscle mass, muscle force, endurance capacity, and intramuscular glycolysis. Furthermore, we analyzed the relationship between skeletal muscle function and intramuscular glycolysis and found that glycolysis-related metabolite regulated protein synthesis by activating intramuscular calcium signaling. These studies highlight the novel role of NAD⁺-related genes in the LH for the regulation of whole-body metabolism and skeletal muscle functions. Our results suggest the importance of NAD⁺ and NAD⁺-related genes in the LH for the pathogenesis of sarcopenia during aging.

Methods: We performed an rs671 genotype-stratified genome-wide association study meta-analysis of alcohol consumption in 175,672 Japanese individuals to explore gene-gene interactions with rs671 behind drinking behavior. Further, to validate the impact of the discovered loci on alcohol-related disease, we conducted a meta-analysis of two esophageal cancer case-control studies.

Results: Three loci (GCKR, KLB, and ADH1B) satisfied the genome-wide significance threshold in wild-type homozygotes (GG), whereas six loci (GCKR, ADH1B, ALDH1B1, ALDH1A1, ALDH2, and GOT2) did so in heterozygotes (GA). Of these, two loci (ALDH2 and GOT2) were novel, and five loci (ADH1B, ALDH1B1, ALDH1A1, ALDH2, and GOT2) showed genome-wide significant interaction with rs671. Of the identified loci, five (GCKR, KLB, ADH1B, ALDH1A1, and ALDH2) were confirmed to be associated with esophageal cancer risk, exhibiting the same direction of association as the rs671 genotype-stratified GWAS.

Conclusions: Our results identify a new genetic architecture associated with alcohol consumption and reveal its potential impact on alcohol-related disease risk. This hypothesis-based genotype-stratified GWAS provides an excellent example of a GWAS conducted using a promising alternative approach that uncovered loci with differential influence on phenotype among genotypes and detected loci whose effects had been indistinct in previous GWASs.

Epilepsy is characterized by recurrent seizures sometimes accompanied by loss of consciousness and visceral  dysfunction. Developmental epileptic encephalopathy (DEE) in infancy and mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) in childhood are often drug-resistant with long-term negative effects on patients' quality of life. The molecular mechanisms underlying epileptogenesis--the process by which the neuronal network acquires the ability to function in a hypersynchronous manner--are largely unknown.

Loss-of-function mutations in the GIRDIN/CCDC88A gene cause DEE in humans¹. Girdin global knockout mice (gKOs) were developed but showed a preweaning lethal phenotype with growth failure², preventing longitudinal analysis. In this study, we developed a novel lifelong feeding regimen (NLFR) tailored to the gKOs' disabilities. Under the NLFR, gKOs survived beyond 1 year and displayed fully penetrant, robust epileptic phenotypes, including generalized tonic-clonic seizures (GTCSs) (averaging eight per day), frequent interictal electroencephalographic spikes (averaging 430 per hour), and progressive deformation of visceral organs. GTCSs preferentially occurred during sleep (NREM). In addition, gKOs exhibited atypical absence seizures and cognitive abnormalities with impaired consciousness. Histologically, bilateral hippocampi in gKOs exhibited congenital cornu-ammonis splitting, granule cell dispersion, and astrogliosis. Furthermore, analysis of conditional knockouts using multiple Cre-deleters identified a defect in the delivery of interneuron precursors from the medial ganglionic eminence into the hippocampal primordium during embryogenesis as a major cause of epileptogenesis (interneuronopathies)³.

In conclusion, we found that gKOs exhibited epileptic phenotypes similar to those observed in human DEE-SWAS and MTLE-HS,identifying interneuronopathy as the central cause of epileptogenesis in mouse Girdin-DEE. The gKO model using an NLFR combines the contradictory properties of high seizure frequency and long-term survivability. We are now trying to detect whole-brain 3D-propagation patterns of GTCSs via advanced imaging or carriers of GTCS propagation via microdialysis combined with rapid analysis techniques using the gKOs.

              Our research goal is to clarify the dynamics of brain networks underling flexible cognitive behaviors and decision-making using non-human primates, i.e, macaque monkeys. Furthermore, we investigate the biological basis of cognitive diversity through multisensory integration.

Our past studies have revealed the neural mechanisms underlying behavioral task-switching, computationally clarified the brain mechanism that simultaneously estimates the observer's own movemen while an object is moving by using maximum likelihood estimation, and also found through decoding analysis that flexible switching between retinal coordinates and external-world-centered coordinates is carried out in the area VIP. Furthermore, our latest study identified that the distinct neural circuits responsible for balancing risk vs. reward-return decision-making with optogenetics, a method that can modulate the activity of specific neurons with light. This study showed the behavioral changes resulting from stimulating neural circuits accumulate over time and have long-term consequences, independent of any stimulus, providing insights into potential mechanisms underlying pathological risk-taking behaviors such as gambling disorders.

Based on these research achievements, we have currently been conducting multispecies empirical comparative research mainly focusing on approaches using non-human primate model animals that have higher-order brains, which will enable us to clarify the evolutionary background of the dynamics of brain neural circuits that lead to the complexity and multidimensionality of decision-making and behavioral choice in animals including humans.

We develop various behavioral paradigms with realistic environment utilizing virtual reality (VR) technology, and perform computational analysis based on large-scale neural activity recordings and neural circuit manipulation applying optogenetics. Through these techniques, we aim to understand the neural dynamics of diverse cognitive behavioral systems from both functional and causal aspects. In particular, we focus on motion systems, spatial navigation systems, balancing system for risk-reward decisions, tracking and avoidance systems, as well as artistic cognition created through flexible integration processing of multisensory inputs and its generating mechanism of impressions and emotions.