Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a healthy individual: WT-iPSC2

AbstractExpanded human T cells from a Japanese healthy male were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, WT-iPSC2, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layer. WT-iPSC2 may be a useful cell resource as a normal control for the comparative study using disease-specific iPSCs

The Japan Monkey Centre Primates Brain Imaging Repository for comparative neuroscience: an archive of digital records including records for endangered species

Advances in magnetic resonance imaging (MRI) and computational analysis technology have enabled comparisons among various primate brains in a three-dimensional electronic format. Results from comparative studies provide information about common features across primates and species-specific features of neuroanatomy. Investigation of various species of non-human primates is important for understanding such features, but the majority of comparative MRI studies have been based on experimental primates, such as common marmoset, macaques, and chimpanzee. A major obstacle has been the lack of a database that includes non-experimental primates’ brain MRIs. To facilitate scientific discoveries in the field of comparative neuroanatomy and brain evolution, we launched a collaborative project to develop an open-resource repository of non-human primate brain images obtained using ex vivo MRI. As an initial open resource, here we release a collection of structural MRI and diffusion tensor images obtained from 12 species: pygmy marmoset, owl monkey, white-fronted capuchin, crab-eating macaque, Japanese macaque, bonnet macaque, toque macaque, Sykes’ monkey, red-tailed monkey, Schmidt’s guenon, de Brazza’s guenon, and lar gibbon. Sixteen postmortem brain samples from the 12 species, stored in the Japan Monkey Centre (JMC), were scanned using a 9.4-T MRI scanner and made available through the JMC collaborative research program (http://www.j-monkey.jp/BIR/index_e.html). The expected significant contributions of the JMC Primates Brain Imaging Repository include (1) resources for comparative neuroscience research, (2) preservation of various primate brains, including those of endangered species, in a permanent digital form, (3) resources with higher resolution for identifying neuroanatomical features, compared to previous MRI atlases, (4) resources for optimizing methods of scanning large fixed brains, and (5) references for veterinary neuroradiology. User-initiated research projects beyond these contributions are also anticipated

Bioluminescent system for dynamic imaging of cell and animal behavior

AbstractThe current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies

The Japan Monkey Centre Primates Brain Imaging Repository of high-resolution postmortem magnetic resonance imaging: the second phase of the archive of digital records

超高磁場MRIで見る霊長類「全脳」神経回路の多様性 --分野横断型の霊長類脳標本画像リポジトリ：ヒト脳と精神・神経疾患の理解を加速する国際研究基盤--. 京都大学プレスリリース. 2023-05-22.A comparison of neuroanatomical features of the brain between humans and our evolutionary relatives, nonhuman primates, is key to understanding the human brain system and the neural basis of mental and neurological disorders. Although most comparative MRI studies of human and nonhuman primate brains have been based on brains of primates that had been used as subjects in experiments, it is essential to investigate various species of nonhuman primates in order to elucidate and interpret the diversity of neuroanatomy features among humans and nonhuman primates. To develop a research platform for this purpose, it is necessary to harmonize the scientific contributions of studies with the standards of animal ethics, animal welfare, and the conservation of brain information for long-term continuation of the field. In previous research, we first developed a gated data-repository of anatomical images obtained using 9.4-T ex vivo MRI of postmortem brain samples from 12 nonhuman primate species, and which are stored at the Japan Monkey Centre. In the present study, as a second phase, we released a collection of T2-weighted images and diffusion tensor images obtained in nine species: white-throated capuchin, Bolivian squirrel monkey, stump-tailed macaque, Tibet monkey, Sykes’ monkey, Assamese macaque, pig-tailed macaque, crested macaque, and chimpanzee. Our image repository should facilitate scientific discoveries in the field of comparative neuroscience. This repository can also promote animal ethics and animal welfare in experiments with nonhuman primate models by optimizing methods for in vivo and ex vivo MRI scanning of brains and supporting veterinary neuroradiological education. In addition, the repository is expected to contribute to conservation, preserving information about the brains of various primates, including endangered species, in a permanent digital form

Visualization of nerve fibers around the carotid bifurcation with use of a 9.4 Tesla microscopic magnetic resonance diffusion tensor imaging with tractography

BACKGROUND: Precise imaging of nerves have been challenging in the head and neck region, mainly due to low spatial resolution. Here, we investigated how nerves in the head and neck region could be visualized using an ultra-high magnetic field MR system. METHODS: We used formol-carbol-fixed human cadaveric necks and obtained MR diffusion tensor images (DTIs) using a 9.4 Tesla (T) ultra-high magnetic field MR system. Afterward, we prepared tissue sections and checked the anatomic relationships between the neurons and the carotid artery in order to confirm that the visualized fibers are indeed neuron fibers. RESULTS: We were able to identify nerves, including the vagus nerve, the hypoglossal nerve, and the spinal-accessory nerve. Hematoxylin-eosin stained histological sections confirmed neuron fibers in the same anatomic position. CONCLUSION: This technique has the feasibility to be applied for a more accurate anatomic understanding, maybe even close to a histological level

Gene Targeting and Subsequent Site-Specific Transgenesis at the beta-actin (ACTB) Locus in Common Marmoset Embryonic Stem Cells

Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the beta-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice

Cadherin-7 Regulates Mossy Fiber Connectivity in the Cerebellum

Summary: To establish highly precise patterns of neural connectivity, developing axons must stop growing at their appropriate destinations and specifically synapse with target cells. However, the molecular mechanisms governing these sequential steps remain poorly understood. Here, we demonstrate that cadherin-7 (Cdh7) plays a dual role in axonal growth termination and specific synapse formation during the development of the cerebellar mossy fiber circuit. Cdh7 is expressed in mossy fiber pontine nucleus (PN) neurons and their target cerebellar granule neurons during synaptogenesis and selectively mediates synapse formation between those neurons. Additionally, Cdh7 presented by mature granule neurons diminishes the growth potential of PN axons. Furthermore, knockdown of Cdh7 in PN neurons in vivo severely impairs the connectivity of PN axons in the developing cerebellum. These findings reveal a mechanism by which a single bifunctional cell-surface receptor orchestrates precise wiring by regulating axonal growth potential and synaptic specificity. : Spatiotemporally orchestrated mechanisms for terminating axonal growth and forming specific synapses are essential in establishing precise neural connectivity. Kuwako et al. demonstrate that cadherin-7 critically regulates the connectivity of the cerebellar mossy fiber circuit by mediating growth inhibition of innervating mossy fiber axons and selective synapse formation between mossy fiber neurons and cerebellar granule neurons. This study reveals that a single cell-surface receptor may play a dual role in axonal growth termination and synaptic specificity to develop precise wiring

α-Synuclein-mediated neurodegeneration in Dementia with Lewy bodies:the pathobiology of a paradox

Dementia with Lewy bodies (DLB) is epitomized by the pathognomonic manifestation of α-synuclein-laden Lewy bodies within selectively vulnerable neurons in the brain. By virtue of prion-like inheritance, the α-synuclein protein inexorably undergoes extensive conformational metamorphoses and culminate in the form of fibrillar polymorphs, instigating calamitous damage to the brain’s neuropsychological networks. This epiphenomenon is nebulous, however, by lingering uncertainty over the quasi “pathogenic” behavior of α-synuclein conformers in DLB pathobiology. Despite numerous attempts, a monolithic “α-synuclein” paradigm that is able to untangle the enigma enshrouding the clinicopathological spectrum of DLB has failed to emanate. In this article, we review conceptual frameworks of α-synuclein dependent cell-autonomous and non-autonomous mechanisms that are likely to facilitate the transneuronal spread of degeneration through the neuraxis. In particular, we describe how the progressive demise of susceptible neurons may evolve from cellular derangements perpetrated by α-synuclein misfolding and aggregation. Where pertinent, we show how these bona fide mechanisms may mutually accentuate α-synuclein-mediated neurodegeneration in the DLB brain

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a healthy individual: WT-iPSC4

AbstractExpanded human T cells from a Japanese healthy male were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, WT-iPSC4, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layer. WT-iPSC4 may be a useful cell resource as a normal control for the comparative study using disease-specific iPSCs

Analysis of RNA metabolism in peripheral WBCs of TDP-43 KI mice identifies novel biomarkers of ALS

AbstractDiagnostic biomarkers for amyotrophic lateral sclerosis (ALS) have yet to be identified. One of the causes of neuronal cell death in neurodegenerative diseases is abnormal RNA metabolism, although the mechanisms by which this occurs are unclear. Detection of abnormal RNA metabolism in white blood cells (WBCs) could lead to a new biomarker of ALS onset. TAR DNA-binding protein 43kDa (TDP-43) is an RNA-binding protein that regulates RNA metabolism. We previously developed a mouse model of ALS that exhibits adult-onset motor dysfunction; these mutant TDP-43 knock in (KI) mice heterozygously express mutant human TDP-43 (A382T or G348C). In the present study, we examined TDP-43 mRNA levels in WBCs of KI mice and found that A382T mutant mRNA is significantly higher than G348C. Our results suggest that each mutant TDP-43 induces distinct RNA metabolism, and that the expression of total TDP-43 alone in WBC is not suitable as an ALS biomarker. To identify additional candidates, we focused on survival and apoptosis-related factors and examined their mRNA metabolism in WBCs. mRNA levels of both Smn1 and Naip5 correlated with TDP-43 levels and also differed between A382T and G348C. Together, TDP-43 and these factors may enable detection of abnormalities in individual ALS pathologies