Biology subjects

Iwata, N.

Publications and source records attributed to Iwata, N..

9 recordsLinked to original sources

bioRxiv2026

Trem2 R47H mutation shows mild, but functionally divergent alterations in microglial phenotypes compared to Trem2 deficiency in aged AppNL-F knock-in mice

The TREM2 R47H variant increases the risk of Alzheimers disease (AD), yet its functional impact in aged mouse models remains incompletely understood. We generated a humanized Trem2 R47H knock-in (KI) line on the AppNL-F background and compared it with a Trem2 knockout (KO) line to assess the degree of TREM2 functional impairment. Accumulation of amyloid {beta} 42 and formation of dystrophic neurites were increased in Trem2 KO mice but not in Trem2 R47H KI mice at 18 or 24 months. qPCR and transcriptomic analyses revealed Trem2 KO mice showed deficits in upregulation of microglial genes while Trem2 R47H KI mice showed a response similar to control mice. Differential gene expression analysis identified altered expressions of genes responsible for ER stress/unfolded protein response and intracellular signalling in Trem2 R47H KI mice. Among the differentially expressed genes, Pmel and Gpnmb were or tended to be downregulated in Trem2 R47H KI as well as in Trem2 KO mice indicating their involvement in AD pathogenesis. These results clearly indicate that the TREM2 R47H variant confers a mild, rather than null, effect on microglial alterations during AD development and that Trem2 R47H KI mice should be used to understand pathological mechanism elicited by TREM2. Further identification and characterization of genes differentially expressed in Trem2 R47H KI mice will provide important insights into how the TREM2 risk variant modulates Alzheimers disease-related pathology. HighlightsO_LIExon2-humanized Trem2 R47H knock-in mice are established, which will serve as a platform to study the role of TREM2 in Alzheimers disease development. C_LIO_LITrem2 knockout mice exhibit deficits in clearance of highly aggregated A{beta}42, suppression of dystrophic neurites and regulation of microglial genes in AppNL-F mice, whereas Trem2 R47H knock-in mice do not. C_LIO_LIRNA-seq reveals transcriptional profiles of Trem2 R47H knock-in mice C_LIO_LIqPCR confirms that Gpnmb and Pmel are or tended to be downregulated in Trem2 R47H knock-in mice. C_LIO_LIFindings demonstrate that TREM2 R47H is hypomorphic rather than loss of function. C_LI

neuroscience↗

bioRxiv2024

Efficient production of functional proaerolysin from E.coli

Proaerolysin is a bacterial toxin produced by Aeromonas hydrophila that specifically binds to GPI-anchored proteins on the plasma membrane, creating transmembrane pores that lead to cell death within a few hours. Leveraging this unique property, proaerolysin is widely used in diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH), a disease caused by somatic mutations in the PIGA gene, which is involved in the biosynthesis of GPI anchors. Additionally, proaerolysin serves as a counter-selection agent in genetic manipulations. Although bacterial expression and purification of proaerolysin have been previously reported, yields were low due to the absence of internal disulfide bonds, which are crucial for protein stability. Here, we demonstrate that using the Shuffle E. coli strain, which facilitates the formation of disulfide bonds in the cytoplasm, significantly improves the solubility and proper folding of proaerolysin. We achieved a high yield of proaerolysin, approximately 3 mg from a 50 ml bacterial culture, with a purity of over 99%. The functionality of recombinant proaerolysin was confirmed by testing in mouse embryonic stem cells (mESCs), demonstrating that this high-yield production method offers a reliable and cost-effective source of functional proaerolysin for a wide range of biotechnological applications.

biochemistry↗

bioRxiv2024

Dual expansion routes likely underlie the present-day population structure in a Parnassius butterfly across the Japanese Archipelago

The Japanese Archipelago, comprising a series of isolated yet interconnected islands, had been geographically separated from the Eurasian continent. The linear topography presents a unique biogeographic context for dispersing organisms from the continent. In this study, we utilized mitochondrial DNA (mtDNA) and single nucleotide polymorphism (SNP) variation to elucidate the dispersal history of the Japanese clouded butterfly Parnassius glacialis across the Japanese Archipelago, including North Island (Hokkaido), Main Island (Honshu) and Shikoku Island. Our analysis of mtDNA (COI, COII) at 1192 base pairs (bps) revealed 49 haplotypes and identified three distinct haplotype groups in the network. These groups correspond geographically to East Japan, West Japan, and Chugoku-Shikoku. The Chugoku-Shikoku group is the most ancient lineage. Interestingly, the Chugoku-Shikoku lineage showed a closer network connection to the East Japan lineage than the geographically proximate West Japan lineage. Divergence time estimates suggest that the Chugoku-Shikoku lineage diverged from the continental P. glacialis approximately 3.05 million years ago (MYA). Subsequently, from the Chugoku-Shikoku lineage, the East Japan and West Japan lineages diverged around 1.05 MYA, with the subsequent divergence between the East and West Japan lineages occurring at approximately 0.62 MYA. Based on the 3067 SNP genotypes, population structure analysis revealed five distinct genetic structures within the Japanese Archipelago, indicating geographical differentiation. From the analyses by mtDNA and SNP variations, four primary genetic barriers were identified: between Hokkaido and Honshu, between East and Central Japan, within the Kansai region, and within the Chugoku region. The former three lines corresponded to the Blakiston Line, the Itoigawa-Shizuoka Tectonic Line, and Lake Biwa, respectively. These findings suggest that P. glacialis diverged from the continental P. glacialis and expanded its range across the Japanese Archipelago via the North and South routes, establishing its current distribution.

molecular biology↗

bioRxiv2024

Metabolic resistance of Abeta3pE-42, epitope of the anti-Alzheimer therapeutic antibody, donanemab.

The amyloid {beta} peptide (A{beta}) starting with pyroglutamate (pE) at position 3 and ending at position 42 (A{beta}3pE-42) is a dominant species that accumulates in the Alzheimers disease (AD) brain. Consistently, a therapeutic antibody raised against this species, donanemab, has been shown to be effective in recent clinical trials. While the primary A{beta} species produced physiologically is A{beta}1-40/42, an explanation for how and why this physiological A{beta} is converted to the pathological form has remained elusive. The conversion of A{beta}1-42 to A{beta}3pE-42 is likely to take place after deposition of A{beta}1-42 given that A{beta}3pE-42 plaques arise significantly later than A{beta}1-42 deposition in the brains of single App knock-in and APP-transgenic mice. Here, we present experimental evidence that accounts for the aging-associated A{beta}3pE-42 deposition: [1] A{beta}3pE-42 is metabolically more stable than other A{beta}X-42 species; [2] Deficiency of neprilysin (NEP), the major A{beta}-degrading enzyme, induces a relatively selective deposition of A{beta}3pE-42 in APP-Tg mice. [3] A{beta}3pE-42 deposition always colocalizes with cored plaques in both APP-Tg and App knock-in mouse brains; [4] A{beta}3E-42, an immediate precursor of A{beta}3pE-42, as well as A{beta}2A-42 and A{beta}4F-42 are more short-lived than A{beta}1-42 in vivo, indicating that simple N-terminal truncation that can arise enzymatically or spontaneously makes A{beta}X-42 easier to catabolize. Consistently, newly generated knock-in mice, AppNL-({Delta}DA)-F and AppNL-({Delta}DA)-Q-F, showed no detectable A{beta} pathology even after aging, indicating that the A{beta}3E-42 and A{beta}3Q-42 species are extremely labile to the in vivo catabolic system and that the E/Q cyclase activity present in mouse brain is insufficient for A{beta}3pE-42 generation. In addition, a deficiency of NEP facilitated A{beta}3pE-42 deposition. Of note, we identified a trace amount of A{beta}3pE-42 and its immediate precursor, A{beta}3E-42, in the insoluble fraction of NEP-deficient APP-Tg mouse brains. A{beta}3pE-42 is thus likely to be a probabilistic by-product of A{beta}1-42 metabolism that selectively accumulates over a long-time range of brain aging. It is likely produced in the solid state or at the solid-liquid interface. Our findings suggest that anti-A{beta} therapies will probably be most effective if given before A{beta}3pE-42 deposition takes place.

neuroscience↗

bioRxiv2021

Neprilysin-sensitive amyloidogenic Aβ versus IDE-sensitive soluble Aβ: a probable mechanistic cause for sporadic Alzheimer's disease

Neprilysin (NEP) and insulin-degrading enzyme (IDE) are considered the two major catabolic enzymes that degrade amyloid {beta} peptide (A{beta}), the primary cause of Alzheimers disease (AD). However, their roles in A{beta} metabolism in vivo have never been compared in an impartial and side-by-side manner. Here, we crossbred single App knock-in mice with NEP (Mme) KO mice and with IDE (Ide) KO mice to generate double mutants that were analyzed for their biochemical and A{beta} pathology properties. We found that NEP is responsible for the metabolism of amyloidogenic insoluble A{beta} whereas IDE affects soluble A{beta}. A deficiency of NEP, but not of IDE, augmented the formation of A{beta} plaques, dystrophic neurites, and astrocytic and microglial activation, all of which are key pathological events in the development of AD. In addition, a deficiency of NEP had no significant impact on the levels of various neuropeptides (somatostatin, substance P, cholecystokinin, and neuropeptide Y), well known to be in vitro substrates for NEP, presumably because NEP is expressed in secretory vesicles and on the presynaptic membranes of excitatory neurons while most if not all neuropeptides are secreted from inhibitory neurons. This argues against the concern that NEP up-regulation for treatment of preclinical AD would reduce the levels of these neuropeptides. These findings indicate that NEP relatively selectively degrades A{beta} in the brain. Whereas familial AD (FAD) is unambiguously caused by an increased anabolism of A{beta}, and of A{beta} 42 and A{beta} 43 in particular, the anabolism of A{beta} appears unaffected before its deposition in the brain that subsequently leads to the onset of sporadic AD (SAD). These observations thus suggest that NEP-sensitive amyloidogenic A{beta} likely plays a primary pathogenic role in the etiology of SAD. Our findings are consistent with the aging-dependent decline of NEP expression in human brain and with recent genome-wide association studies (GWAS) indicating that variants of the gene encoding NEP (MME) are associated with the risk of SAD development. Taken together, our results imply that the aging-associated decrease in NEP expression is a primary cause of SAD and could thus be a target for the treatment of preclinical AD once other factors such as apolipoprotein E genotypes have also been considered.

neuroscience↗

bioRxiv2021

Field Potential Image Classification of Pacemaker Micro-coordination in the Intestine of the Mouse

The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an expanding or a migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of expanding and migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals.

physiology↗

bioRxiv2020

Sex-Dependent Shared and Non-Shared Genetic Architecture Across Mood and Psychotic Disorders

BACKGROUNDSex differences in incidence and/or presentation of schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BIP) are pervasive. Previous evidence for shared genetic risk and sex differences in brain abnormalities across disorders suggest possible shared sex-dependent genetic risk. METHODSWe conducted the largest to date genome-wide genotype-by-sex (GxS) interaction of risk for these disorders, using 85,735 cases (33,403 SCZ, 19,924 BIP, 32,408 MDD) and 109,946 controls from the Psychiatric Genomics Consortium (PGC) and iPSYCH. RESULTSAcross disorders, genome-wide significant SNP-by-sex interaction was detected for a locus encompassing NKAIN2 (rs117780815; p=3.2x10-8), that interacts with sodium/potassium-transporting ATPase enzymes implicating neuronal excitability. Three additional loci showed evidence (p<1x10-6) for cross-disorder GxS interaction (rs7302529, p=1.6x10-7; rs73033497, p=8.8x10-7; rs7914279, p=6.4x10-7) implicating various functions. Gene-based analyses identified GxS interaction across disorders (p=8.97x10-7) with transcriptional inhibitor SLTM. Most significant in SCZ was a MOCOS gene locus (rs11665282; p=1.5x10-7), implicating vascular endothelial cells. Secondary analysis of the PGC-SCZ dataset detected an interaction (rs13265509; p=1.1x10-7) in a locus containing IDO2, a kynurenine pathway enzyme with immunoregulatory functions implicated in SCZ, BIP, and MDD. Pathway enrichment analysis detected significant GxS of genes regulating vascular endothelial growth factor (VEGF) receptor signaling in MDD (pFDR<0.05). CONCLUSIONSIn the largest genome-wide GxS analysis of mood and psychotic disorders to date, there was substantial genetic overlap between the sexes. However, significant sex-dependent effects were enriched for genes related to neuronal development, immune and vascular functions across and within SCZ, BIP, and MDD at the variant, gene, and pathway enrichment levels.

genetics↗

bioRxiv2020

Promoter activity-based case-control association study on SLC6A4 highlighting hypermethylation and altered amygdala volume in male patients with schizophrenia

Associations between altered DNA methylation of the serotonin transporter (5-HTT)-encoding gene SLC6A4 and early life adversity, mood and anxiety disorders, and amygdala reactivity have been reported. However, few studies have examined epigenetic alterations of SLC6A4 in schizophrenia (SZ). We examined CpG sites of SLC6A4, whose DNA methylation levels have been reported to be altered in bipolar disorder, using three independent cohorts of patients with SZ and age-matched controls. We found significant hypermethylation of a CpG site in SLC6A4 in male patients with SZ in all three cohorts. We showed that chronic administration of risperidone did not affect the DNA methylation status at this CpG site using common marmosets, and that in vitro DNA methylation at this CpG site diminished the promoter activity of SLC6A4. We then genotyped the 5-HTT-linked polymorphic region (5-HTTLPR) and investigated the relationship among 5-HTTLPR, DNA methylation, and amygdala volume using brain imaging data. We found that patients harboring low-activity 5-HTTLPR alleles showed hypermethylation and they showed a negative correlation between DNA methylation levels and left amygdala volumes. These results suggest that hypermethylation of the CpG site in SLC6A4 is involved in the pathophysiology of SZ, especially in male patients harboring low-activity 5-HTTLPR alleles.

genetics↗

bioRxiv2019

Large scale genome-wide association study in a Japanese population identified 45 novel susceptibility loci for 22 diseases

INTRODUCTORY PARAGRAPHThe overwhelming majority of participants in current genetic studies are of European ancestry1-3, limiting our genetic understanding of complex disease in non-European populations. To address this, we aimed to elucidate polygenic disease biology in the East Asian population by conducting a genome-wide association study (GWAS) with 212,453 Japanese individuals across 42 diseases. We detected 383 independent signals in 331 loci for 30 diseases, among which 45 loci were novel (P < 5 x 10-8). Compared with known variants, novel variants have lower frequency in European populations but comparable frequency in East Asian populations, suggesting the advantage of this study in discovering these novel variants. Three novel signals were in linkage disequilibrium (r2 > 0.6) with missense variants which are monomorphic in European populations (1000 Genomes Project) including rs11235604(p.R220W of ATG16L2, a autophagy-related gene) associated with coronary artery disease. We further investigated enrichment of heritability within 2,868 annotations of genome-wide transcription factor occupancy, andidentified 378 significant enrichments across nine diseases (FDR < 0.05) (e.g. NF-{kappa}B for immune-related diseases). This large-scale GWAS in a Japanese population provides insights into the etiology of common complex diseases and highlights the importance of performing GWAS in non-European populations.

genetics↗