The nucleolus is a biomolecular condensate maintained by multivalent intermolecular interactions. However, the physicochemical interactions that preserve the nucleolar integrity remain poorly understood. We established an isolated nucleolus assay to investigate the mechanisms that maintain nucleolar integrity. Increasing the ionic strength or adding basic amino acids disrupted the nucleolar organization, whereas 1,6-hexanediol and RNase treatment had little effect, highlighting the major role of electrostatic interactions. Arginine-rich dipeptide repeat proteins associated with C9orf72-linked neurodegeneration caused greater disruption than free arginine, depending on the repeat length and concentration. The assay further revealed that unlike the wild-type HMGB1, a disease-associated HMGB1 mutant resisted arginine-induced release from the nucleoli. These findings identify electrostatic interactions as major determinants of nucleolar integrity and provide a platform for investigating disease-associated alterations in nucleolar organization.

Frontotemporal dementia (FTD) is an early onset form of dementia characterized by frontotemporal lobar atrophy accompanied by behavioral, personality, language, and motor deficits. Heterozygous mutations in GRN gene encoding progranulin (PGRN) are the genetic causes of FTD. Since PGRN is a neurotrophic and anti-inflammatory factor, most FTD-related PGRN mutations are thought to cause FTD due to haploinsufficiency. Therefore, therapies that increase PGRN levels by the administration of recombinant PGRN or viral vectors are attracting attention as an approach to the treatment of FTD. However, the mechanisms underlying the neuroprotective effects of PGRN remain unclear. To investigate the neuroprotective mechanisms of PGRN in vivo, we generated human PGRN transgenic (Tg) mice using the CAG promoter. Unexpectedly, mice overexpressing wild-type human PGRN showed a shortened lifespan and cerebellar dysfunction, including the loss of Purkinje cells. Furthermore, PGRN Tg mice developed cognitive impairment, gliosis, and lysosomal abnormalities. FTD-causative R432C-PGRN mutant Tg mice also showed FTD-like phenotypes, such as neuronal loss, gliosis, and behavioral deficits. In cultured cells, overexpression of PGRN induced endoplasmic reticulum (ER) stress and apoptotic cell death, suggesting that continuous increases in PGRN expression through viral vectors or genetic manipulation are neurotoxic and that PGRN-replacement therapy may be required to maintain optimal PGRN levels for each neuron type and brain region.

Arginine-rich dipeptide repeat proteins (R-DPRs), produced from hexanucleotide repeat expansions via repeat-associated non-AUG translation, are linked to neurodegenerative disorders. While novel, potentially pathogenic R-DPRs have been identified, their intracellular signaling mechanisms remain unclear. To explore their biological roles, we examined R-DPR liquid-liquid phase separation behavior in vitro and their impact on nucleolar function. We found that the spacer hydrophobicity is a key determinant of R-DPR phase separation. Specifically, R-DPRs with hydrophobic spacers formed aggregates with RNA and recombinant nucleophosmin in vitro, inducing nucleolar stress and impairing ribosomal RNA synthesis in cells. These findings provide insights into the mechanisms by which R-DPRs exert nucleolar stress, advancing our understanding of their pathological roles.

Macromolecular crowding is a fundamental property of the intracellular environment that influences protein folding, enzymatic activity, and phase behavior. Disruptions to the homeostasis of macromolecular crowding can drive pathological processes, such as aberrant liquid-liquid phase separation and protein aggregation, which are central features of several neurodegenerative diseases. However, tools for quantifying crowding and aggregation remain limited. Here, we describe moxCRONOS, a Förster resonance energy transfer (FRET)-based biosensor that enables the quantitative measurement of macromolecular crowding and protein condensation. moxCRONOS retains the optical properties of the original CRONOS sensor but offers enhanced stability in oxidative environments, such as within the endoplasmic reticulum or under sodium arsenite treatment, allowing for direct comparison of crowding levels across organelles regardless of redox conditions. Moreover, when fused to dipeptide repeat proteins associated with C9ORF72-linked neurodegeneration, moxCRONOS detects aggregation-prone states-especially in cells expressing glycine-alanine (GA) repeats. Using fluorescence-activated cell sorting, we achieved sensitive and quantitative detection of heterogeneous high-FRET cell populations containing GA aggregates. FRET signal intensity increased upon treatment with a molecular crowding agent or a proteasome inhibitor. These findings establish moxCRONOS as a versatile biosensor for investigating both physiological macromolecular crowding and pathological protein aggregation, with significant potential for disease modeling and therapeutic screening.

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron-specific degenerative disease, and frontotemporal dementia (FTD) is another neurodegenerative disease that causes atrophy of the frontal and temporal lobes. There are pathological and genetical overlap between ALS and FTD regarding their causative genes and the presence of ubiquitin- and TDP-43-positive inclusion bodies. However, the pathogenesis of ALS and FTD remains insufficiently characterized. CHCHD10 (C10), a familial ALS/FTD-causative gene, is expressed in mitochondria and is thought to be involved in the regulation of mitochondrial functions, although the mechanism underlying the C10 mutation-mediated onset of ALS/FTLD remains unknown. In this study, we generated C10-expressing adenoviruses to unravel the mechanism underlying cell death caused by C10 mutations. The results showed that overexpression of wild-type C10 or a C10 mutant induced cell death in parallel with increase in the transcription factor C/EBP homologous protein (CHOP) expression, the indicator of mitochondrial unfolded protein response. Importantly, the overexpression of the C10 mutant caused higher-level cell death and expression of CHOP than wild-type C10. These results suggest that the familial ALS/FTLD-linked mutation of C10 enhances motor neuron toxicity possibly by exaggerated mitochondrial stress.

A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Unconventional translation of the expanded repetitive sequence generates five dipeptide repeat proteins (DPRs) and these DPRs can cause neurotoxicity. The molecular mechanism underlying the DPR-linked neurotoxicity is under investigation. In this study, we show that poly-proline-arginine DPR (poly-PR), the most neurotoxic DPR in vitro, binds to adenosine deaminase acting on RNA (ADAR) 1 and ADAR2 and inhibits their RNA editing activity. We further show that poly-PR impairs cellular stress response that is mediated by ADAR1. These results together suggest that the poly-PR-mediated inhibition of the ADAR activity contributes to C9-ALS/FTD-linked neurotoxicity.

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Unconventional translation of the hexanucleotide repeat expansion generates five dipeptide repeat proteins (DPRs). The molecular mechanism underlying the DPR-linked neurotoxicity is under investigation. In this study, using cell-based models, we show that poly-proline-arginine DPR (poly-PR), the most neurotoxic DPR in vitro, binds to adenosine deaminase acting on RNA (ADAR)1p110 and ADAR2 and inhibits their RNA editing activity. We further show that poly-PR impairs cellular stress response that is mediated by ADAR1p110. These results together suggest that the poly-PR-mediated inhibition of the ADAR activity contributes to C9-ALS/FTD-linked neurotoxicity.

A GGGGCC hexanucleotide repeat expansion in the C9ORF72gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, bound to and up-regulated nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 caused neurotoxicity. We also show that the poly-PR caused neurotoxicity by modulating the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulated NEAT1 expression and induced neurotoxicity. These results suggest that the dysfunction of paraspeckles is linked to the poly-PR- and TDP-43-mediated neurotoxicity.

A GGGGCC repeat expansion in the C9ORF72gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat proteins. Although it has been reported that dipeptide repeat proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we show that the expression of proline-arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity. The poly-PR-induced neurotoxicity is restored by the acceleration of ribosomal RNA synthesis. This result suggests that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. Furthermore, we show that poly-PR interacts with multiple DEAD-box RNA helicases and inhibits the function of at least one of them, and that the reduction in the levels of some RNA helicases results in both the decrease in ribosomal RNA levels and the increase in neuronal cell death. Altogether, these results suggest that poly-PR causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce five dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, binds to and up-regulates nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 causes neurotoxicity. We also show that the poly-PR modulates the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulates NEAT1 expression and induces neurotoxicity. These results suggest that the increase in the paraspeckle formation may be involved in the poly-PR- and TDP-43-mediated neurotoxicity.

A computer simulation application on pharmacokinetics, which we developed using a software, named "Stella®", has been successfully used for the virtual training of pharmacokinetics at multiple medical schools. The training course using Stella® has encouraged the medical students to optimize drug administration for individual patients on the computers. Importantly, the virtual training is free of any concern on human and animal ethics. The simulation application has been freely provided for medical schools without any restrictions and charge. For many years, it has been under constant version-upgrade in response to updates of the operating systems (OS) of personal computers or the software. Very recently, major updates of the OS and the software, and the emergence of tablet- and smartphones-type computers have been prompting us to perform a major revision of the simulation application. Here, we introduce the new version of the "web-based" simulation application that is available through any device including personal computers, tablets, and smartphones irrespective of the OSs (Microsoft Windows and Macintosh, Android, and iOS), without any extra charge unless the modification is required. We believe that the new-version of web-based simulation application will be useful not only for medical, nursing and pharmacy students, but also for medical workers who need to simulate drug pharmacokinetics on the computers before they administer drugs to the patients.

Amyotrophic lateral sclerosis (ALS) is a motor neuron-specific neurodegenerative disease and frontotemporal dementia (FTD) is a neurodegenerative disease with young-onset dementia. Accumulating evidence indicates that they have common clinical and pathologic features. Several mutations of the CHCHD10 (C10) gene have been found to cause ALS/FTD. Wild-type C10 is localized at mitochondria and physiologically involved in the regulation of mitochondrial function. It has been previously shown that a mutant C10 induces mitochondrial dysfunction such as the reduction in ATPase production that has been hypothesized to be closely linked to the ALS/FTD onset. In the present study, we have investigated the molecular mechanism underlying neuronal cell death caused by a mutant C10, S59L-C10. We have found that the adenovirus-medicated overexpression of wild-type C10 and S59L-C10 induces cell death in vitro. As expected, S59L-C10-induced cell death was more prominent than that induced by wild-type C10. This result suggests that S59L-C10-induced cell death may be caused by the gain-of-toxic mechanism. We have further characterized the pathway in detail underlying the S59L-C10-induced cell death.

A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat (DPR) proteins. Although it has been reported that DPR proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we have first confirmed that proline-arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity and found that the poly-PR-induced neurotoxicity is repressed by the acceleration of ribosomal RNA synthesis. These results suggest that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. We have further identified DEAD-box RNA helicases as poly-PR-binding proteins, the functions of which are inhibited by poly-PR. The enforced reduction in the expression of DEAD-box RNA helicases causes impairment of ribosome biogenesis and neuronal cell death. These results together suggest that poly-PR causes neurotoxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

Heterogeneous nuclear ribonucleoprotein (hnRNP)A1, a member of the hnRNP family, is involved in a variety of RNA metabolisms. The hnRNPA1 expression is altered in some human diseases and mutations of the hnRNPA1 gene cause amyotrophic lateral sclerosis and multisystem proteinopathy. It has been therefore assumed that the dysregulation of hnRNPA1 is linked to the pathogenesis of the diseases. However, the mechanism underlying the regulation of the hnRNPA1 expression remains unknown. In this study, using cell-based models, we have found that hnRNPA1 negatively regulates its own mRNA expression by inhibiting the intron10 splicing of hnRNPA1 pre-mRNA. This mechanism likely serves as an autoregulation of the hnRNPA1 expression. We have also found that a low-grade excess of hnRNPA1 expression causes cytotoxicity by activating the mitochondrial apoptosis pathway. Collectively, these data suggest that the level of hnRNPA1 is strictly controlled to be within a certain range by the mRNA autoregulation in the physiological condition so that the cytotoxicity-causative alteration of hnRNPA1 expression does not take place.

A common genetic variation in the transmembrane protein 106B (TMEM106B) gene has been suggested to be a risk factor for frontotemporal lobar degeneration (FTLD) with inclusions of transactive response DNA-binding protein-43 (TDP-43) (FTLD-TDP), the most common pathological subtype in FTLD. Furthermore, previous studies have shown that TMEM106B levels are up-regulated in the brains of FTLD-TDP patients, although the significance of this finding remains unknown. In this study, we show that the overexpression of TMEM106B and its N-terminal fragments induces cell death, enhances oxidative stress-induced cytotoxicity, and causes the cleavage of TDP-43, which represents TDP-43 pathology, using cell-based models. TMEM106B-induced death is mediated by the caspase-dependent mitochondrial cell death pathways and possibly by the lysosomal cell death pathway. These findings suggest that the up-regulation of TMEM106B may increase the risk of FTLD by directly causing neurotoxicity and a pathological phenotype linked to FTLD-TDP.

医学教育分野別認証取得に向けた臨床実習時間の拡大に伴い，基礎医学の講義・実習時間数が減少する．これに起因する学習時間の不足を補うためには，学生自身が自ら学ぶ姿勢へと導く教育方法の改善が必要であり，そのための有力な手段のひとつとして，認証取得の一必要条件ともなっているe-learningシステムが考えられる．今回，東京医科大学に導入されているe-learningシステムを使用し，血中濃度モニタリングシミュレーション実習について事前学習の導入を試みた．本報告では，実習終了後のアンケート調査ならびに提出レポートの内容，その他を分析することにより，e-learningシステムを用いた事前学習の効果について検討した．その結果，e-learningシステムを用いた事前学習は学生の実習内容に対する理解度を有意に高め，授業時間内に円滑な薬理学シミュレーション実習を遂行することに寄与することが明らかとなった．この結果は，e-learningシステムを今後広く学生の自主学習を助ける手段として活用することの妥当性を支持している．

Dysregulation of transactive response DNA-binding protein-43 (TDP-43) is thought to be linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 normally localizes in the nucleus but its main localization shifts to the cytoplasm in most affected cells of ALS and FTLD patients. It is not yet known whether nuclear or cytoplasmic TDP-43 is responsible for TDP-43-induced neurotoxicity. In this study, we show that nuclear TDP-43 causes TDP-43 neurotoxicity. DNA/RNA-binding and dimerization of TDP-43 are both essential for TDP-43-induced cell death. Moreover, endogenous heterogeneous nuclear ribonucleoprotein-U (hnRNP-U) binds to TDP-43 and knocking-down of hnRNP-U induces neurotoxicity, whereas overexpression of hnRNP-U or hnRNP-A2 inhibits TDP-43-induced neurotoxicity. In addition, hnRNP-U inhibits TDP-43-mediated alterations in splicing of POLDIP3 mRNA. Altogether, these results suggest that nuclear TDP-43 becomes neurotoxic by escaping from the inhibitory regulation by hnRNPs.