Requirement of the Ca2+ channel ß2 subunit for sympathetic PKA phosphorylation.

Facilitation of cardiac function in response to signals from the sympathetic nervous system is initiated by the phosphorylation of L-type voltage-dependent Ca2+ channels (VDCCs) by protein kinase A (PKA), which in turn is activated by ß-adrenoceptors. Among the five subunits (α1, ß, α2/δ, and γ) of VDCCs, the α1 subunit and the family of ß subunits are substrates for PKA-catalyzed phosphorylation; however, the subunit responsible for ß-adrenergic augmentation of Ca2+ channel function has yet to be specifically identified. Here we show that the VDCC ß2 subunit is required for PKA phosphorylation upon sympathetic acceleration. In mice with ß2 subunit-null mutations, cardiac muscle contraction in response to isoproterenol was reduced and there was no significant increase in Ca2+ channel currents upon PKA-dependent phosphorylation. These findings indicate that within the sympathetic nervous system the ß2 subunit of VDCCs is required for physiological PKA-dependent channel phosphorylation.

Ganglioside Synthase Knockout Reduces Prion Disease Incubation Time in Mouse Models.

Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid-containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft-associated proteins. To investigate the role of gangliosides in the pathogenesis of prion diseases, we performed intracerebral transmission study of a scrapie prion strain Chandler and a Gerstmann-Sträussler-Scheinker syndrome prion strain Fukuoka-1 using various knockout mouse strains ablated with ganglioside synthase gene (ie, GD2/GM2 synthase, GD3 synthase, or GM3 synthase). After challenge with the Chandler strain, GD2/GM2 synthase knockout mice showed 20% reduction of incubation time, reduced prion protein deposition in the brain with attenuated glial reactions, and reduced localization of prion proteins to lipid rafts. These results raise the possibility that the gangliosides may have an important role in prion disease pathogenesis by affecting the localization of prion proteins to lipid rafts.

Contribution of rat embryonic stem cells to xenogeneic chimeras in blastocyst or 8-cell embryo injection and aggregation.

The ultimate goal of regenerative medicine is the transplantation of a target organ generated by the patient's own cells. Recently, a method of organ generation using pluripotent stem cells (PSCs) and blastocyst complementation was reported. This approach is based on chimeric animal generation using an early embryo and PSCs, and the contribution of PSCs to the target organ is key to the method's success. However, the contribution rate of PSCs in target organs generated by different chimeric animal generation methods remains unknown. In this study, we used 8-cell embryo aggregation, 8-cell embryo injection, and blastocyst injection to generate interspecies chimeric mice using rat embryonic stem (ES) cells and then investigated the differences in the contribution rate of the rat ES cells. The rate of chimeric mouse generation was the highest using blastocyst injection, followed in order by 8-cell embryo injection and 8-cell embryo aggregation. However, the contribution rate of rat ES cells was the highest in chimeric neonates generated by 8-cell embryo injection, and the difference was statistically significant in the liver. Live functionality was confirmed by analyzing the expression of rat hepatocyte-derived drug-metabolizing enzyme. Collectively, these findings indicate that the 8-cell embryo injection method is the most suitable for generation of PSC-derived organs via chimeric animal generation, particularly for the liver.

Ubiquitin-Specific Protease 2 Modulates the Lipopolysaccharide-Elicited Expression of Proinflammatory Cytokines in Macrophage-like HL-60 Cells.

We investigated the regulatory roles of USP2 in mRNA accumulation of proinflammatory cytokines in macrophage-like cells after stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Human macrophage-like HL-60 cells, mouse macrophage-like J774.1 cells, and mouse peritoneal macrophages demonstrated negative feedback to USP2 mRNA levels after LPS stimulation, suggesting that USP2 plays a significant role in LPS-stimulated macrophages. USP2 knockdown (KD) by short hairpin RNA in HL-60 cells promoted the accumulation of transcripts for 25 of 104 cytokines after LPS stimulation. In contrast, limited induction of cytokines was observed in cells forcibly expressing the longer splice variant of USP2 (USP2A), or in peritoneal macrophages isolated from Usp2a transgenic mice. An ubiquitin isopeptidase-deficient USP2A mutant failed to suppress LPS-induced cytokine expression, suggesting that protein ubiquitination contributes to USP2-mediated cytokine repression. Although USP2 deficiency did not accelerate TNF receptor-associated factor (TRAF) 6-nuclear factor-κB (NF-κB) signaling, it increased the DNA binding ratio of the octamer binding transcription factor (Oct)-1 to Oct-2 in TNF, CXCL8, CCL4, and IL6 promoters. USP2 decreased nuclear Oct-2 protein levels in addition to decreasing the polyubiquitination of Oct-1. In summary, USP2 modulates proinflammatory cytokine induction, possibly through modification of Oct proteins, in macrophages following TLR4 activation.

Modified autonomic regulation in mice mutated in the ß4 subunit of the lh/lh calcium channel.

Genetic analyses have revealed an important association between P/Q-type calcium channel activities and hereditary neurological disorders. The P/Q-type channels are composed principally of heterologous multimeric subunits including CaV2.1 and CaVß4. Of these, the ß4 subunit is thought to play a significant role in channel physiology, because a mouse line mutant in that subunit (the lethargic mouse: lh) exhibits a severe ataxic phenotype. The aim of the present study was to elucidate the physiological importance of the ß4 subunit. ECG analysis showed that the T wave was high in 8-week-old lh mutants; this may be associated with hyperkalemia. Upon pharmacological ECG analysis, 2-3-week-old lh mutants exhibited reduced responses to a ß-blocker and a muscarinic receptor antagonist. Analysis of heart rate variability revealed that the R-R interval was unstable in lh mutants and that both the low- and high-frequency components had increased in extent, indicating that the tonus of both the sympathetic and parasympathetic nervous systems was modified. Thus, our present study revealed that the ß4 subunit played a significant role in regulation of sympathetic and parasympathetic nerve activities.

A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.

Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histological analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal-appearing p53-positive epithelium that are similar to 'p53 signatures' in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non-invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and wild-type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease.

Colony-stimulating factor-1 signaling suppresses renal crystal formation.

We recently reported evidence suggesting that migrating macrophages (Mϕs) eliminate renal crystals in hyperoxaluric mice. Mϕs can be inflammatory (M1) or anti-inflammatory (M2), and colony-stimulating factor-1 (CSF-1) mediates polarization to the M2Mϕ phenotype. M2Mϕs promote renal tissue repair and regeneration, but it is not clear whether these cells are involved in suppressing renal crystal formation. We investigated the role of M2Mϕs in renal crystal formation during hyperoxaluria using CSF-1-deficient mice, which lack M2Mϕs. Compared with wild-type mice, CSF-1-deficient mice had significantly higher amounts of renal calcium oxalate crystal deposition. Treatment with recombinant human CSF-1 increased the expression of M2-related genes and markedly decreased the number of renal crystals in both CSF-1-deficient and wild-type mice. Flow cytometry of sorted renal Mϕs showed that CSF-1 deficiency resulted in a smaller population of CD11b(+)F4/80(+)CD163(+)CD206(hi) cells, which represent M2-like Mϕs. Additionally, transfusion of M2Mϕs into CSF-1-deficient mice suppressed renal crystal deposition. In vitro phagocytosis assays with calcium oxalate monohydrate crystals showed a higher rate of crystal phagocytosis by M2-polarized Mϕs than M1-polarized Mϕs or renal tubular cells. Gene array profiling showed that CSF-1 deficiency resulted in disordered M2- and stone-related gene expressions. Collectively, our results provide compelling evidence for a suppressive role of CSF-1 signaling in renal crystal formation.

Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo.

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1+/- nor Chk2-/- mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1+/-Chk2-/- and Chk1+/-Chk2+/- mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.

Ubiquitin-specific protease 2-69 in macrophages potentially modulates metainflammation.

Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2-69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/FABP4 and PAI-1/SERPINE1 genes were increased by 4- and 1.8-fold, respectively, after short hairpin RNA-mediated knockdown (KD) of the USP2 gene, and such expression was alleviated by overexpression of USP2-69 in human myeloid cell lines. Supernatants derived from USP2-KD cells induced IL6 (∼6-fold) and SAA3 (∼15-fold) in 3T3-L1 adipocytes to suggest the anti-inflammatory properties of USP2. In addition, we observed a 30% decrease in the number of macrophages in mesenteric adipose tissue derived from USP2-69 transgenic mice fed a high-fat diet for 14 wk compared with that in their C57BL/6 littermates (P<0.01), which was consistent with a ∼40% decrease in transcription of aP2 and PAI-1. The aP2 locus exhibited elevated chromatin accessibility (>2.1-fold), methylation of histone H3 lysine 4 (>4.5-fold), and acetylation of histone H4 (>2.5-fold) in USP2-KD cells. Transfection of isopeptidase-mutated USP2-69 did not alter chromatin conformation on the aP2 locus in USP2-KD cells. Our results suggest that USP2-69 suppresses meta-inflammatory molecules involved in the development of type-2 diabetes.

A novel transgenic mouse model carrying human Tribbles related protein 3 (TRB3) gene and its site specific phenotype.

Tribbles related protein 3 (TRB3) pseudokinase plays a crucial role in cell proliferation, migration and morphogenesis during development. In our recent study, an introduction of human TRB3 gene into mouse mammary tumor cells caused an increase of proliferation of tumor cells and their nuclear size. In the current study, to examine whether this gene causes de novo morphological changes in a specific organ site we have developed a novel variation of the transgenic mouse model that conditionally expresses human TRB3 (hTRB3) gene using Cre-recombinase (Cre)/loxP recombination system. By injecting hTRB3 transgene construct into pronuclei of mouse embryo, we eventually obtained four hTRB3 mice. The gene expression was controlled by infection of adenovirus-expressing Cre via the tail vein of hTRB3 mouse. In Cre-mediated hTRB3 mouse, expression of the hTRB3 protein was detected in the cytoplasm of hepatocytes in the liver. Expression of this protein was also seen in lymphocytes in the spleen, glomerular endothelial cells, and epithelial cells of collecting duct of the kidney. In hepatocytes of the hTRB3 mouse, nuclear size was significantly greater than that of the wild type mouse, indicating that hTRB3 can play a role at least in part in hepatic morphogenesis. The present animal model may provide a system for evaluation of de novo morphological changes induced by a specific transgene in a specific organ site.

Targeted disruption of fad24, a regulator of adipogenesis, causes pre-implantation embryonic lethality due to the growth defect at the blastocyst stage.

In previous studies, we identified a novel gene, factor for adipocyte differentiation 24 (fad24), which plays an important role during the early stages of adipogenesis in mouse 3T3-L1 cells. Moreover, overexpression of fad24 increased the number of smaller adipocytes in white adipose tissue and improved glucose metabolic activity in mice, thus indicating that fad24 functions as a regulator of adipogenesis in vivo. However, the physiological roles of fad24 in vivo are largely unknown. In this study, we attempted to generate fad24-deficient mice by gene targeting. No fad24-null mutants were recovered after embryonic day 9.5 (E9.5). Although fad24-null embryos were detected in an expected Mendelian ratio of genotypes at E3.5, none of the homozygous mutants developed into blastocysts. In vitro culture experiments revealed that fad24-null embryos develop normally to the morula stage but acquire growth defects during subsequent stages. The number of nuclei decreased in fad24-deficient morulae compared with that in wild-type ones. These results strongly suggested that fad24 is essential for pre-implantation in embryonic development, particularly for the progression to the blastocyst stage.

Exclusive expression of VMAT2 in noradrenergic neurons increases viability of homozygous VMAT2 knockout mice.

The vesicular monoamine transporter 2 (VMAT2) translocates monoamine neurotransmitters from the neuronal cytoplasm into synaptic vesicles. Since VMAT2-/- mice die within a few days of birth, it is difficult to analyze the detailed VMAT2 functions using these mice. In this study, we generated human VMAT2 transgenic mice that expressed VMAT2 in noradrenergic neurons with the aim to rescue the lethality of VMAT2 deletion. The expression of human VMAT2 in noradrenergic neurons extended the life of VMAT2-/- mice for up to three weeks, and these mice showed severe growth deficiency compared with VMAT2+/+ mice. These results may indicate that VMAT2 expressed in noradrenergic neurons has crucial roles in survival during the first several weeks after birth, and VMAT2 functions in other monoaminergic systems could be required for further extended survival. Although VMAT2 rescue in noradrenergic neurons did not eliminate the increased morbidity and lethality associated with VMAT2 deletion, the extension of the lifespan in VMAT2 transgenic mice will enable behavioral, pharmacological and pathophysiological studies of VMAT2 function.

A simple, dual direct expression plasmid system in prokaryotic and mammalian cells.

We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic (Escherichia coli) and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on E. coli for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.

Enhanced ß-adrenergic response in mice with dominant-negative expression of the PKD2L1 channel.

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure in humans. Among the various PKD-related molecules, PKD2L1 forms cation channels, but its physiological importance is obscure. In the present study, we established a transgenic mouse line by overexpressing the dominant-negative form of the mouse PKD2L1 gene (i.e., lacking the pore-forming domain). The resulting PKD2L1del-Tg mice exhibited supraventricular premature contraction, as well as enhanced sensitivity to ß-adrenergic stimulation and unstable R-R intervals in electrocardiography. During spontaneous atrial contraction, PKD2L1del-Tg atria showed enhanced sensitivity to isoproterenol, norepinephrine, and epinephrine. Action potential recording revealed a shortened action potential duration in PKD2L1del-Tg atria in response to isoproterenol. These findings indicated increased adrenergic sensitivity in PKD2L1del-Tg mice, suggesting that PKD2L1 is involved in sympathetic regulation.

Impairment of neuropsychological behaviors in ganglioside GM3-knockout mice.

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. Although its expression is tightly controlled during early embryo development and postnatal development and maturation in the brain, the physiological role of ganglioside GM3 in the regulation of neuronal functions has not been elucidated. In the present study, we examined motor activity, cognitive and emotional behaviors, and drug administration in juvenile GM3-knockout (GM3-KO) mice. GM3-KO male and female mice showed hyperactivity in the motor activity test, Y-maze test, and elevated plus maze test. In the Y-maze test, there was significantly less spontaneous alternation behavior in GM3-KO male mice than in wild-type mice. In the elevated plus maze test, the amount of time spent on the open arms by GM3-KO male mice was significantly higher than that of sex-matched wild-type mice. In contrast, there was no significant difference between GM3-KO and wild-type female mice in these tests. Thus, juvenile GM3-KO mice show gender-specific phenotypes resembling attention-deficit hyperactivity disorder (ADHD), namely hyperactivity, reduced attention, and increased impulsive behaviors. However, administration of methylphenidate hydrochloride (MPH) did not ameliorate hyperactivity in either male or female GM3-KO mice. Although these data demonstrate the involvement of ganglioside GM3 in ADHD and the ineffectiveness of MPH, the first-choice psychostimulant for ADHD medication, our studies indicate that juvenile GM3-KO mice are a useful tool for neuropsychological studies.

Rap signaling is crucial for the competence of IL-7 response and the development of B-lineage cells.

Rap family GTPases consist of multiple members with substantial functional redundancy. With the use of transgenic mice conditionally expressing a bona fide dominant-negative Rap1 mutant, Rap1A17, capable of inhibiting the activation of all Rap family members in B-lineage cells (mb.1-Rap1A17 Tg), we demonstrate that these mice show a defective generation of pre-B cells in bone marrow, resulting in a significant diminution of peripheral mainstream B cells. The effect is attributed to the impaired survival and expansion of B-lineage progenitors in response to IL-7, despite normal IL-7Ralpha expression. The pre-B cells from mb.1-Rap1A17 Tg mice showed a significantly reduced expression of c-myc and E2A, and the competence of IL-7 response was restored by the transduction of c-myc, but not by constitutively active (CA) Stat5a, CA PI3K-p100, or bcl-2. The residual follicular B cells with complete Cre-mediated recombination proliferated normally in response to B-cell receptor stimulation and showed efficient germinal center reaction in vivo. These results show that endogenous Rap signaling plays a crucial role selectively in B-lineage cell development by sustaining the competence for IL-7 response, whereas it is dispensable for mature B-cell function.

Subventricular zone-derived neural progenitor cells migrate along a blood vessel scaffold toward the post-stroke striatum.

The subventricular zone (SVZ) of the adult brain contains neural stem cells that have the capacity to regenerate new neurons after various insults. Brain ischemia causes damage to brain tissue and induces neural regeneration together with angiogenesis. We previously reported that, after ischemic injury in mice, SVZ-derived neural progenitor cells (NPCs) migrate into the striatum, and these NPCs are frequently associated with blood vessels in the regenerating brain tissue. Here we studied the role of blood vessels during the neural regeneration in more detail. BrdU administration experiments revealed that newly generated NPCs were associated with both newly formed and pre-existing blood vessels in the ischemic striatum, suggesting that the angiogenic environment is not essential for the neuron-blood vessel interaction. To observe migrating NPCs and blood vessels simultaneously in damaged brain tissue, we performed live imaging of cultured brain slices after ischemic injury. In this system, we virally labeled SVZ-derived NPCs in Flk1-EGFP knock-in mice in which the blood vessels are labeled with EGFP. Our results provide direct evidence that SVZ-derived NPCs migrate along blood vessels from the SVZ toward the ischemic region of the striatum. The leading process of the migrating NPCs was closely associated with blood vessels, suggesting that this interaction provides directional guidance to the NPCs. These findings suggest that blood vessels play an important role as a scaffold for NPCs migration toward the damaged brain region.

TP53/p53-FBXO22-TFEB controls basal autophagy to govern hormesis.

Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy. Mild stress-activated TP53 transcriptionally induced FBXO22, which in turn ubiquitinated KDM4B (lysine-specific demethylase 4B) complexed with MYC-NCOR1 suppressors for degradation, leading to transcriptional induction of TFEB. Upregulation of autophagy-related genes by increased TFEB dramatically enhanced autophagic activity and cell survival upon following a severe stressor. Mitogen-induced AKT1 activation counteracted this process through the phosphorylation of KDM4B, which inhibited FBXO22-mediated ubiquitination. Additionally, fbxo22-/- mice died within 10 h of birth, and their mouse embryonic fibroblasts (MEFs) showed a lowered basal autophagy, whereas FBXO22-overexpressing mice were resistant to chemotherapy. Taken together, these results suggest that TP53 upregulates basal autophagy through the FBXO22-TFEB axis, which governs the hormetic effect in chemotherapy.Abbreviations: BBC3/PUMA: BCL2 binding component 3; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; ChIP-seq: chromatin immunoprecipitation followed by sequencing; DDB2: damage specific DNA binding protein 2; DRAM: DNA damage regulated autophagy modulator; ESR/ER: estrogen receptor 1; FMD: fasting mimicking diet; HCQ: hydroxychloroquine; KDM4B: lysine-specific demethylase 4B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; NCOR1: nuclear receptor corepressor 1; SCF: SKP1-CUL-F-box protein; SQSTM1: sequestosome 1; TFEB: transcription factor EB.

Two mouse models carrying truncating mutations in Magel2 show distinct phenotypes.

Schaaf-Yang syndrome (SYS) is a neurodevelopmental disorder caused by truncating variants in the paternal allele of MAGEL2, located in the Prader-Willi critical region, 15q11-q13. Although the phenotypes of SYS overlap those of Prader-Willi syndrome (PWS), including neonatal hypotonia, feeding problems, and developmental delay/intellectual disability, SYS patients show autism spectrum disorder and joint contractures, which are atypical phenotypes for PWS. Therefore, we hypothesized that the truncated Magel2 protein could potentially produce gain-of-function toxic effects. To test the hypothesis, we generated two engineered mouse models; one, an overexpression model that expressed the N-terminal region of Magel2 that was FLAG tagged with a strong ubiquitous promoter, and another, a genome-edited model that carried a truncating variant in Magel2 generated using the CRISPR/Cas9 system. In the overexpression model, all transgenic mice died in the fetal or neonatal period indicating embryonic or neonatal lethality of the transgene. Therefore, overexpression of the truncated Magel2 could show toxic effects. In the genome-edited model, we generated a mouse model carrying a frameshift variant (c.1690_1924del; p(Glu564Serfs*130)) in Magel2. Model mice carrying the frameshift variant in the paternal or maternal allele of Magel2 were termed Magel2P:fs and Magel2M:fs, respectively. The imprinted expression and spatial distribution of truncating Magel2 transcripts in the brain were maintained. Although neonatal Magel2P:fs mice were lighter than wildtype littermates, Magel2P:fs males and females weighed the same as their wildtype littermates by eight and four weeks of age, respectively. Collectively, the overexpression mouse model may recapitulate fetal or neonatal death, which are the severest phenotypes for SYS. In contrast, the genome-edited mouse model maintains genomic imprinting and distribution of truncated Magel2 transcripts in the brain, but only partially recapitulates SYS phenotypes. Therefore, our results imply that simple gain-of-function toxic effects may not explain the patho-mechanism of SYS, but rather suggest a range of effects due to Magel2 variants as in human SYS patients.

A domain responsible for spontaneous conversion of bank vole prion protein.

Bank vole is a small rodent that shows high susceptibility to infection with diverse prion strains. To determine whether the increased susceptibility of bank voles to prion diseases can be attributed to the intrinsic nature of bank vole prion protein (PrP) or to host factors other than PrP, we produced transgenic mice overexpressing bank vole PrP. These transgenic mice spontaneously developed neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain. Then, we produced transgenic mice overexpressing chimeric mouse/bank vole PrP, which differs from mouse PrP only at two residues located at the C-terminus, to determine the minimum essential domain for the induction of spontaneous generation of abnormal PrP. These transgenic mice also developed spontaneous neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain. In addition, knock-in mice expressing bank vole PrP at the same level as that of wild-type mice did not develop spontaneous disease but showed high susceptibility to infection with diverse prion strains, similarly to bank voles. Taken together, these findings show that bank vole PrP has a high propensity for the conformational conversion both in spontaneous disease and in prion infection, probably due to the characteristic structural properties of the C-terminal domain.