Biology subjects

Nakatani, K.

Publications and source records attributed to Nakatani, K..

11 recordsLinked to original sources

bioRxiv2025

Interventionally targeting somatic CAG expansions can be a rapid disease-modifying therapeutic avenue: Preclinical evidence.

Huntington disease (HD) is caused by inherited CAG expansions, which continue expanding somatically in affected brain regions to hasten disease onset and progression. Therapeutically diminishing somatic expansions is expected to be clinically beneficial. However, it is not known if interventionally modifying somatic CAG expansions will actually modify in vivo clinically-relevant phenotypes, what the therapeutic window is, or which phenotypes will be altered. Here we show that acute (6-week) delivery of the contraction-inducing slipped-CAG DNA ligand naphthyridine-azaquinolone to young (4-week-old) (CAG)120 HD mice, induces contractions throughout brain regions, improves motor function (locomotion, balance, coordination, muscle strength), molecular disease landmarks (mHTT aggregates, nuclear envelope morphology, nucleocytoplasmic mRNA transport, transcriptomic dysregulation, neuroinflammation), and neurodegeneration. Beneficial effects of modifying somatic expansions were also evident in muscle and blood, where blood CAG instability correlated with brain instability and blood serum had diminished levels of neurofilament light (a biomarker for neurodegeneration) -- offering blood as having elements of target engagement and efficacy. These data support that targeting somatic repeat expansions can be a rapid disease-modifying therapeutic avenue for HD and possibly other repeat expansion diseases. Our findings support an etiologic pathway interconnected to somatic CAG expansions that will inform the design of clinical trials expecting clinical benefit by modulating somatic expansions.

genetics↗

bioRxiv2025

Characterizing lipoprotein profiles in coronary atherosclerosis development through quantitative lipidomics and proteomics approaches

Lipoproteins, which are synthesized in hepatic cells and secreted into the blood, play an important role in lipid transport. The quality of lipoproteins, including small dense low-density lipoproteins and the cholesterol efflux capacity of high-density lipoproteins, influences cardiovascular outcomes, although the relationship between lipid changes and lipoprotein quality remains unclear. This study characterized lipid molecules and apolipoproteins in lipoprotein particles during the development of coronary atherosclerosis in myocardial infarction-prone rabbits. Using quantitative lipidomics and proteomics techniques, the lipid composition in lipoprotein particles was evaluated by normalizing lipidomics data with lipoprotein particle numbers estimated by apolipoprotein B-100 concentration. While apolipoproteins and common risk factors for abnormal lipid metabolism, including cholesterol and triacylglycerol, did not change across stages of coronary atherosclerosis, ceramide molecules in lipoprotein particles were significantly associated with early stage progression. This study provides a perspective on the role of lipid composition in the progression of coronary atherosclerosis.

bioengineering↗

bioRxiv2023

Antisense RNA C9orf72 Hexanucleotide Repeat Associated With Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Forms A Triplex-Like Structure and Binds Small Synthetic Ligand

The abnormal expansion of GGGGCC/CCCCGG hexanucleotide repeats (HR) in C9orf72 is associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Structural polymorphisms of HR result in the multifactorial pathomechanism of ALS/FTD. Consequently, many ongoing studies are focused at developing therapies targeting pathogenic HR RNA. One of them involves small molecules blocking the sequestration of important proteins, preventing the formation of toxic nuclear foci. However, rational design of potential therapeutics is hindered by limited number of structural studies of RNA-ligand complexes. We determined the crystal structure of antisense HR RNA in complex with ANP77 ligand and in the free form. HR RNA folds into a triplex structure composed of four RNA chains. ANP77 interacted with two neighboring single-stranded cytosines to form pseudo-canonical base pairs by adopting sandwich-like conformation and adjusting the position of its naphthyridine units to the helical twist of the RNA. In the unliganded structure, the cytosines formed a peculiar triplex i-motif, assembled by trans C*C+ pair and a third cytosine located at the Hoogsteen edge of the C*C+ pair. These results extend our knowledge of the structural polymorphisms of HR and can be used for the rational design of small molecules targeting disease-related RNAs.

molecular biology↗

bioRxiv2023

Low albumin status accompanies multi-layered immunosuppressive phenotypes in metastatic breast cancer patients

Low albumin status is prevalent in advanced cancer patients, but the pathophysiology associated with this anomaly remains largely unexplored. To address this, we aim to search correlations of albumin levels with the transcriptome against peripheral blood mononuclear cells and the plasma metabolome within the same patients having metastatic breast cancers. We confirm that metastatic breast cancer patients exhibit low albumin levels in varying degrees without prominent systemic inflammation. Our data demonstrate that low albumin levels correlate with transcriptome signatures indicative of "neutrophil activation and T-cell down-regulation," an immunosuppressive phenotype. We also find that immunoregulatory metabolites, such as arginine, are reduced in plasma in an albumin-correlated manner, further corroborating systemic immunosuppression. These results are verified using a mouse model of breast cancer. We conclude that low albumin status in metastatic breast cancer patients accompanies immunosuppressive phenotypes, which is likely unfavorable for anti-cancer immunotherapy and thus can be a cause of unsuccessful treatment outcomes.

cancer biology↗

bioRxiv2023

Inhibition of OCT4 Binding at the MYCN Locus Induces Neuroblastoma Cell Death Accompanied by Downregulation of Transcripts with High-Open Reading Frame Dominance

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether other transcription factors contribute to NB cell survival. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN activity. Differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with high-ORF dominance were significantly associated with genes whose high expression is associated with a poor prognosis of NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the human-specific OCT4-MYCN positive feedback loop may serve as an effective therapeutic strategy for MYCN-amplified NB. Contribution to the fieldNeuroblastoma (NB) is a childhood tumor. Amplification of MYCN is frequently observed in high-risk NBs and is linked to a poor prognosis. Multiple transcription factors, including OCT4, MYCN, CTCF, and p53, regulate MYCN expression by binding to the MYCN locus. This study investigated the contribution of these transcription factors in NB cell survival. We used CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in MYCN-amplified NB cell lines. We found that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, reduces MYCN activity and induces NB cell death. A detailed investigation of the molecular mechanisms of cell death revealed that the downregulated transcripts after suppressed MYCN activity were associated with high-open reading frame (ORF) dominance scores, which are associated with translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Reduced expression of these splicing factors altered the PKM mRNA splicing accompanied by the induction of p53-caspase-2-MDM2-mediated cell death. These findings suggest that disrupting the human-specific OCT4-MYCN positive feedback loop may serve as a promising therapeutic strategy for MYCN-amplified NB.

cancer biology↗

bioRxiv2023

Global changes in open reading frame dominance of RNAs during cancer initiation and progression

Cancer cells express unique RNA transcripts; however, the factors determining their translation have remained unclear. We recently developed open reading frame (ORF) dominance as a measure that correlates with coding potential of RNAs. Upon calculating the ORF dominance of cancer-specific transcripts across 24 human tumor types, 14 exhibited significantly higher ORF dominance in cancer than in normal tissues. In organoid-based mouse genetic models, ORF dominance increased with carcinogenesis. Gene ontology analysis revealed that gene sets with increased ORF dominance were associated with cell proliferation, while those with decreased ORF dominance were linked to DNA damage response. Translatome analyses demonstrated that elevated ORF dominance during carcinogenesis resulted in higher translation frequencies of ribosome-bound RNAs. As cancer progressed, ORF dominance showed that the boundary between coding and noncoding transcripts became blurred prior to distant metastasis, indicating decreased proliferative cell populations and increased generation of RNA isoforms that potentially translate neoantigens before the development of metastatic tumors. These findings suggest that cancer evolution leads to dynamic changes in ORF dominance, resulting in global translational alterations in transcriptomes.

cancer biology↗

bioRxiv2023

Wide scope analysis of bioactive lipids, including steroids, bile acids, and polyunsaturated fatty acid metabolites, in human plasma by LC/MS/MS

Quantitative information on blood metabolites has the potential to be utilized in medical strategies such as early disease detection and prevention. Monitoring of bioactive lipids, such as steroids, bile acids, and polyunsaturated fatty acid (PUFA) metabolites, could be a valuable indicator for health status. However, a method for simultaneous measurement of these bioactive lipids has not been reported at present. Here, we report a liquid chromatography tandem mass spectrometry (LC/MS/MS) method that can simultaneously measure more than 140 bioactive lipids, including steroids, bile acids, and PUFA metabolites, from human plasma, and a sample preparation method for these targets. Protein removal in methanol precipitation and purification operations of bioactive lipids by solid-phase extraction improved the recovery of targeted compounds in human plasma samples, demonstrating the importance of sample preparation methods in a wide range of bioactive lipid analyses. Using the developed method, we measured plasma from healthy human volunteers and confirmed the presence of bioactive lipid molecules associated with sex differences and circadian rhythms. The practical bioactive lipid analysis method developed is expected to be applied to health monitoring and disease biomarker discovery for precision medicine.

systems biology↗

bioRxiv2022

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

neuroscience↗

bioRxiv2021

FAN1 nuclease processes and pauses on disease-associated slipped-DNA repeats: Mechanism against repeat expansions

FAN1 nuclease is a modifier of repeat expansion diseases, including Huntingtons disease (HD), fragile X syndrome, and autism. The age of HD onset correlates with ongoing inchworm-like repeat expansions (1-3 CAG units/event) in HD brains, and is regulated by three modifiers: The first two, repeat tract length and purity exert their effects by enhancing and slowing CAG expansions, respectively, by affecting the formation of slipped-DNAs -- mutagenic intermediates of instability; which are processed to expansions by the third modifiers, DNA repair proteins. FAN1 protects against hyper-expansions of repeats, by unknown mechanisms. We show FAN1, through iterative cycles bound, dimerized and cleaved slipped-DNAs, yielding striking patterns of distinct exo-nuclease pauses along slip-outs; 5'-C{downarrow}A{downarrow}GC{downarrow}A{downarrow}G-3' and 5'-C{downarrow}T{downarrow}G{downarrow}C{downarrow}T{downarrow}G-3'. The transcriptionally-displaced CAG strand was excised slower than its complementary CTG strand, required A*A and T*T mismatches, as fully-paired hairpins arrested excision progression, while disease-delaying CAA interruptions further slowed FAN1 excision. In contrast, endo-nucleolytic cleavage was insensitive to slip-outs. Rare FAN1 variants were found in autism individuals with CGG/CCG repeat expansions. Excision of CGG/CCG slip-outs were similarly excised, with CGG being slower than CCG. The slip-out specific ligand, Naphthyridine-Azaquinolone, shown to induce contractions of expanded repeats in cells, required FAN1 for its effect, and protected slip-outs from FAN1s exo- but not endo-nucleolytic digestion. FAN1s inchworm pausing of slip-out excision is suited to minimize incremental expansions and modulating disease onset.

genetics↗

bioRxiv2021

Potentially translated sequences determine protein-coding potential of RNAs in cellular organisms

Recent studies have identified numerous RNAs that are functionally both coding and noncoding. However, the sequence characteristics that determine bifunctionality remain largely unknown. In this study, we developed and tested a potentially translated island (PTI) score, defined as the occupancy of the longest open reading frame (ORF) among all putative ORFs. We found that this score correlated with translation, including noncoding RNAs. In bacteria and archaea, coding and noncoding transcripts had narrow distributions of high and low PTI scores, respectively, whereas those of eukaryotes showed relatively broader distributions, with considerable overlap between coding and noncoding transcripts. The extent of overlap positively and negatively correlated with the mutation rates of genomes and effective population sizes of species, respectively. These overlaps were significantly increased in threatened species. In macroevolution, the appearance of the nucleus and multicellularity seem to have influenced the overlap of PTI score distributions, so that the probability of the existence of bifunctional RNAs is increased in eukaryotes. In mammalian testes, we observed an enrichment of noncoding RNAs with high PTI scores, which are candidates for bifunctional RNAs. These results suggest that the decrease in population size and the emergence of testes in eukaryotic multicellular organisms allow for the stable existence of bifunctional RNAs, consequently increasing the probability of the birth of novel coding and non-coding RNAs.

evolutionary biology↗

bioRxiv2020

Small molecule targeting r(UGGAA)n disrupts RNA foci and alleviates disease phenotype in Drosophila model

Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here we report our discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA)n repeat RNAs in spinocerebellar ataxia type 31 (SCA31). Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarified the mode of binding that recognizes four guanines in UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)n repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA-protein interactions resulting in the suppression of nSBs-mediated splicing event. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates disease phenotype induced by toxic r(UGGAA)n repeat RNA. These studies demonstrated that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases.

biochemistry↗