A missense mutation in the Hspa8 gene encoding heat shock cognate protein 70 causes neuroaxonal dystrophy in rats.

Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.

A novel Kit mutant rat enables hematopoietic stem cell engraftment without irradiation.

Hematopoietic stem cell (HSC) transplantation is extensively studied in mouse models, but their limited scale presents challenges for effective engraftment and comprehensive evaluations. Rats, owing to their larger size and anatomical similarity to humans, offer a promising alternative. In this study, we establish a rat model with the KitV834M mutation, mirroring KitW41 mice often used in KIT signaling and HSC research. KitV834M rats are viable and fertile, displaying anemia and mast cell depletion similar to KitW41 mice. The colony-forming unit assay revealed that the KitV834M mutation leads to reduced proliferation and loss of or decreased pluripotency of hematopoietic stem and progenitor cells (HSPCs), resulting in diminished competitive repopulating capacity of KitV834M HSPCs in competitive transplantation assays. Importantly, KitV834M rats support donor rat-HSC engraftment without irradiation. Leveraging the larger scale of this rat model enhances our understanding of HSC biology and transplantation dynamics, potentially advancing our knowledge in this field.

Relative stereochemical determination of the C61-C83 fragment of symbiodinolide using a stereodivergent synthetic approach.

Structural determination is required in the use of marine natural products to create novel drugs and drug leads in medicinal chemistry. Symbiodinolide, which is a polyol marine natural product with a molecular weight of 2860, increases the intracellular Ca2+ concentration and exhibits inhibitory activity against cyclooxygenase-1. Seventy percent of the structure of symbiodinolide has been stereochemically clarified. Herein, we report the elucidation of the relative configuration of the C61-C83 fragment, which is among the remaining thirty percent, using a stereodivergent synthetic strategy. We first assigned the relative configuration of the C61-C74 fragment. Two candidate diastereomers of the C61-C74 fragment were synthesized, and their NMR data were compared with those of the natural product, revealing the relative stereochemistry of this component. We then narrowed down the candidate compounds for the C69-C83 fragment from 16 possible diastereomers by analyzing the NMR data of the natural product, and we thus selected eight candidate diastereomers. Stereodivergent synthesis of the candidates for this fragment and comparison of the NMR data of the natural product and the eight synthetic products resulted in the relative stereostructural clarification of the C69-C83 fragment. These individually determined relative stereochemistries of the C61-C74 and C69-C83 fragments were connected via the common C69-C73 tetrahydropyran moiety of the fragments. Finally, the relative configuration of the C61-C83 fragment of symbiodinolide was determined. The stereodivergent synthetic approach used in this study can be extended to the stereochemical determination of other fragments of symbiodinolide.

A high-quality severe combined immunodeficiency (SCID) rat bioresource.

Immunodeficient animals are valuable models for the engraftment of exogenous tissues; they are widely used in many fields, including the creation of humanized animal models, as well as regenerative medicine and oncology. Compared with mice, laboratory rats have a larger body size and can more easily undergo transplantation of various tissues and organs. Considering the absence of high-quality resources of immunodeficient rats, we used the CRISPR/Cas9 genome editing system to knock out the interleukin-2 receptor gamma chain gene (Il2rg) in F344/Jcl rats-alone or together with recombination activating gene 2 (Rag2)-to create a high-quality bioresource that researchers can freely use: severe combined immunodeficiency (SCID) rats. We selected one founder rat with frame-shift mutations in both Il2rg (5-bp del) and Rag2 ([1-bp del+2-bp ins]/[7-bp del+2-bp ins]), then conducted mating to establish a line of immunodeficient rats. The immunodeficiency phenotype was preliminarily confirmed by the presence of severe thymic hypoplasia in Il2rg-single knockout (sKO) and Il2rg/Rag2-double knockout (dKO) rats. Assessment of blood cell counts in peripheral blood showed that the white blood cell count was significantly decreased in sKO and dKO rats, while the red blood cell count was unaffected. The decrease in white blood cell count was mainly caused by a decrease in lymphocytes. Furthermore, analyses of lymphocyte populations via flow cytometry showed that the numbers of B cells (CD3- CD45+) and natural killer cells (CD3- CD161+) were markedly reduced in both knockout rats. In contrast, T cells were markedly reduced but showed slightly different results between sKO and dKO rats. Notably, our immunodeficient rats do not exhibit growth retardation or gametogenesis defects. This high-quality SCID rat resource is now managed by the National BioResource Project in Japan. Our SCID rat model has been used in various research fields, demonstrating its importance as a bioresource.

Combi-CRISPR: combination of NHEJ and HDR provides efficient and precise plasmid-based knock-ins in mice and rats.

CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20-33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.

Evaluation of the otolith function using sinusoidal off-vertical axis rotation in patients with benign paroxysmal positional vertigo.

The vestibulo-ocular reflex (VOR) was studied via sinusoidal off-vertical axis rotation (OVAR) to evaluate the otolith function in patients with benign paroxysmal positional vertigo (BPPV). Subjects were sinusoidally rotated with eyes open in complete darkness at frequencies of 0.4 and 0.8 Hz with a maximum angular velocity of 60 degrees s(-1) in earth-vertical axis rotation (EVAR) and OVAR. Twenty-three controls and 24 BPPV patients were investigated. Results showed that VOR gain during OVAR at 0.8 Hz in a 30 degrees nose-up position in BPPV patients was significantly less than the gain during EVAR, whereas the gain was not significantly different between EVAR and OVAR in the controls in each condition. In addition, to examine each type of BPPV, we also investigated whether there were any differences between the patients who suffered from dizziness and those who did not. VOR gain in OVAR of BPPV patients who were suffering from dizziness was significantly less than that of BPPV patients without dizziness. Not only cupulolithiasis or canalolithiasis, but also otolith dysfunction was considered to be the possible origin of BPPV. Because sinusoidal OVAR produced minimal nausea compared to constant velocity OVAR, the stimulation of 0.8 Hz nose-up in sinusoidal OVAR may be used to evaluate otolith function without discomfort for patients.

Flexibility of vestibulo-ocular reflex adaptation to modified visual input in human.

The vestibulo-ocular reflex (VOR) serves to keep images relatively stable on the retina. To maintain appropriate performance and minimize image slip throughout life, VOR is subjected to long-term adaptive regulation by visual input. It has been reported that adaptive changes in VOR gain (eye velocity/head velocity) are evoked either by fitting subjects with magnifying, miniaturizing, or reversing spectacles during normal behavior, or by moving a large visual field in or out of phase relative to the subject's head movement. The changes in VOR gain are frequency selective. Here, we examine the extent of VOR gain flexibility by causing VORs of similar direction to undergo different behavioral gain changes. Nine healthy adults participated in the study, ranging in age from 24 to 38 years (mean: 26 years) and with no history of neurotological symptoms. All subjects were clinically normal according to a screening battery that included combined neurologic and otologic physical examinations. Horizontal and vertical eye positions were recorded by bitemporal DC-coupled electro-oculography (EOG). The subject sat in a rotating chair. The axis of rotation of the body was always earth-vertical, with the interaural axis crossing the axis of rotation of the chair. The head was pointed 20 degrees downwards in all experiments and stabilized in this position using a chin rest. The chair was surrounded by a half-cylindrical optokinetic screen (78 cm in diameter) placed in front of the subject, onto which random dot patterns were projected. Goggles were used to ensure that the subject was in complete darkness during both pre- and postadaptation periods. The chair was rotated sinusoidally at maximum amplitude of 30 degrees or 60 degrees : for 30 degrees the stimulation was at 0.1, 0.2, 0.3, and 0.4 Hz; for 60 degrees it was at 0.1, 0.2, and 0.3 Hz. VOR adaptation was obtained by inducing a retinal slip velocity by short-term alteration of the visual input of the large field; this change was produced by a combination of sinusoidal head rotation and the random dot patterns. In each adaptation session, the sinusoidal head rotation was either at 0.1 or 0.3 Hz and the amplitude was 30 degrees. The random dot pattern was synchronized with sinusoidal head rotation in the same direction (x0 experiment) to make the retinal slip zero, and in the opposite direction to make the retinal slip twofold (x2 experiment). Therefore, a total of four adaptation protocols were tested. The subjects were asked to fixate on a single dot with the eyes straight-ahead in the x0 experiment, and follow it within random dot patterns in the x2 experiment. Each adaptation session lasted for 30 min. Two adaptation experiments were performed on each subject once per day. The averages of VOR gain and phase lag were calculated using Fourier analysis. Seven of the subjects who participated in the x2 adaptation experiment at 0.3 Hz and 30 degrees amplitude showed a steady increase of VOR gain within a couple of trials. One of the remaining two subjects showed a decrease in VOR gain in all the three trials, and the other subject showed a VOR gain increase in three trials and decrease in two trials. In the x2 adaptation experiment at 0.3 Hz and 30 degrees amplitude (peak velocity: 28 degrees /s), the percentage gain change ((post-pre)/pre) was 133% at the same stimulation and 100% at 0.4 Hz (peak velocity: 37 degrees /s). The gain change was 65% at 0.1 Hz and 60 degrees amplitude (peak velocity: 18 degrees /s), and 64% at 0.2 Hz and 60 degrees amplitude (peak velocity: 37 degrees /s). In the x1 adaptation experiment (30 degrees at 0.3 Hz), the percentage gain change was -62% under the same conditions, -50% at 0.1 Hz and 60 degrees amplitude, and -30% at 0.2 Hz and 60 degrees amplitude. No change of VOR gain was observed at other frequencies, and the subject was not adapted at 0.1 Hz and 30 degrees amplitude. The larger VOR gain increase in the x2 adaptation experiment for the faster head angular acceleration leads to the conclusion that the VOR gain mechanism can set vuite similar in terms of the head acceleration.