The corticotropin-releasing hormone (CRH) and its type 1 receptor (CRHR1) play a central role in coordinating the endocrine, autonomic, and behavioral responses to stress. A prerequisite to functionally dissect the complexity of the CRH/CRHR1 system is to unravel the identity of CRHR1-expressing neurons and their connectivities. Therefore, we used a knockin approach to genetically label CRHR1-expressing cells with a tau-lacZ (tZ) reporter gene. The distribution of neurons expressing β-galactosidase in the brain and the relative intensity of labeling is in full accordance with previously described Crhr1 mRNA expression. Combining the microtubule-binding properties of TAU with the Cre-loxP system allowed to direct the β-galactosidase to proximal dendrites, and in particular to axons. Thereby, we were able to visualize projections of CRHR1 neurons such as glutamatergic and dopaminergic afferent connections of the striatum and GABAergic CRHR1-expressing neurons located within its patch compartment. In addition, the tZ reporter gene revealed novel details of CRHR1 expression in the spinal cord, skin, and eye. CRHR1 expression in the retina prompted the identification of a new physiological role of CRHR1 related to the visual system. Besides its reporter properties, this novel CRHR1 allele comprises the possibility to conditionally restore or delete CRHR1 via Flp and Cre recombinase, respectively. Finally, the allele is suitable for further manipulations of the CRHR1 locus by recombinase-mediated cassette exchange. Taken together, this novel mouse allele will significantly facilitate the neuroanatomical analysis of CRHR1 circuits and opens up new avenues to address CRHR1 function in more detail.

βB2-crystallin (gene symbol: Crybb2/CRYBB2) was first described as a structural protein of the ocular lens. This gene, however, is also expressed in several regions of the mammalian brain, although its function in this organ remains entirely unknown. To unravel some aspects of its function in the brain, we combined behavioral, neuroanatomical, and physiological analyses in a novel Crybb2 mouse mutant, O377. Behavioral tests with male O377 mutants revealed altered sensorimotor gating, suggesting modified neuronal functions. Since these mouse mutants also displayed reduced hippocampal size, we concentrated further investigations on the hippocampus. Free intracellular Ca(2+) levels were increased and apoptosis was enhanced in the hippocampus of O377 mutants. Moreover, the expression of the gene encoding calpain 3 (gene symbol Capn3) was elevated and the expression of genes coding for the NMDA receptor subunits was downregulated. Additionally, the number of parvalbumin-positive interneurons was decreased in the hippocampus but not in the cortex of the mutants. High-speed voltage-sensitive dye imaging demonstrated an increased translation of input-to-output neuronal activity in the dentate gyrus of this Crybb2 mutant. These results point to an important function of βB2-crystallin in the hippocampal network. They indicate pleiotropic effects of mutations in the Crybb2 gene, which previously had been considered to be specific to the ocular lens. Moreover, our results are the first to demonstrate that βB2-crystallin has a role in hippocampal function and behavioral phenotypes. This model can now be further explored by future experiments.

Within a mutagenesis screen, we identified the new mouse mutant Aey80 with small eyes; homozygous mutants were not obtained. The aim of the study was its molecular characterization.

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.

Glioblastoma (GBM) is the most common malignant intracranial tumor with a low survival rate. However, only few drugs responsible for GBM therpies, hence new drug development for it is highly required. The natural product Cudraflavone B (CUB) has been reported to potentially kill a variety of tumor cells. Currently, its anit-cancer effect on GBM still remains unknown. Herein, we investigated whether CUB could affect the proliferation and apoptosis of GBM cells to show anti-GBM potential.

Under the label of the German Mouse Clinic (GMC), a concept has been developed and implemented that allows the better understanding of human diseases on the pathophysiological and molecular level. This includes better understanding of the crosstalk between different organs, pleiotropy of genes, and the systemic impact of envirotypes and drugs. In the GMC, experts from various fields of mouse genetics and physiology, in close collaboration with clinicians, work side by side under one roof. The GMC is an open-access platform for the scientific community by providing phenotypic analysis in bilateral collaborations ("bottom-up projects") and as a partner and driver in international large-scale biology projects ("top-down projects"). Furthermore, technology development is a major topic in the GMC. Innovative techniques for primary and secondary screens are developed and implemented into the phenotyping pipelines (e.g., detection of volatile organic compounds, VOCs).

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Mutations in the X chromosomal tRNA 2'‑O‑methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.

Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.

Optic neuritis is a common inflammatory manifestation of multiple sclerosis (MS). In experimental autoimmune encephalomyelitis (EAE), the optic nerve is affected as well. Here, we investigated whether autoimmune inflammation in the optic nerve is distinct from inflammation in other parts of the central nervous system (CNS). In our study, inflammatory infiltrates in the optic nerve and the brain were characterized by a high fraction of Ly6G(+) granulocytes whereas in the spinal cord, macrophage infiltrates were predominant. At the peak of disease, IL-17 mRNA abundance was highest in the optic nerve as compared with other parts of the CNS. The ratio of IL-17 vs IFN-γ producing CD4(+) T cells was higher in the optic nerve and brain than in the spinal cord and more effector CD4(+) T cells were committed to the Th17 transcriptional program in the optic nerve than in the spinal cord. IL-17 producing γδ T cells but rather not Ly6G(+) granulocytes themselves contributed to IL-17 production. Optical coherence tomography (OCT) studies on murine eyes revealed a decline in thickness of the retinal nerve fiber layer (RNFL) and the common layer of ganglion cells and inner plexiform layer (GCL+) after the recovery from motor symptoms indicating that autoimmune inflammation induced a significant atrophy of optic nerve fibers during EAE. Neutralization of IL-17 by treatment with anti-IL-17 antibodies reduced but did not abrogate motor symptoms of EAE. However, RNFL and GCL+ atrophy were completely prevented by blocking IL-17. Thus, the optic nerve compartment is particularly prone to support IL-17 mediated inflammatory responses during CNS autoimmunity and structural integrity of the retina can be preserved by neutralizing IL-17.

GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.

The clinical phenotype of retinal gliosis occurs in different forms; here, we characterize one novel genetic feature, (i.e., signaling via BMP-receptor 1b).

The CNNM/CorC family proteins are Mg2+ transporters that are widely distributed in all domains of life. In bacteria, CorC has been implicated in the survival of pathogenic microorganisms. In humans, CNNM proteins are involved in various biological events, such as body absorption/reabsorption of Mg2+ and genetic disorders. Here, we determined the crystal structure of the Mg2+-bound CorC TM domain dimer. Each protomer has a single Mg2+ binding site with a fully dehydrated Mg2+ ion. The residues at the Mg2+ binding site are strictly conserved in both human CNNM2 and CNNM4, and many of these residues are associated with genetic diseases. Furthermore, we determined the structures of the CorC cytoplasmic region containing its regulatory ATP-binding domain. A combination of structural and functional analyses not only revealed the potential interface between the TM and cytoplasmic domains but also showed that ATP binding is important for the Mg2+ export activity of CorC.

The age-related reduction in the function of osteoblasts plays a central role in the pathogenesis of bone loss and osteoporosis. Collagen synthesis is a primary function of differentiated osteoblasts, however, the mechanisms for age-related changes in collagen synthesis in human osteoblasts remain elusive. We use Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) analysis to exploit the transcriptional profiles of osteoblasts from young and old donors. A panel of collagen members was downregulated in aged osteoblasts, including COL12A1, COL5A1, COL5A3, COL8A1 and COL8A2. Co-expression analysis followed by GO analysis revealed that oxidoreductase activity and kinase activity were inversely correlated with collagen synthesis in osteoblasts. GESA analysis further showed that JNK signaling was upregulated in aged osteoblasts. Consistently, MAP3K4 and MAP4K2, upstream of JNK, were also increased in aged osteoblasts. Moreover, expression levels of MAP3K4 were significantly inversely correlated with levels of the collagen genes. Those transcriptomic results were further verified by examining clinical specimens of osteoporosis by immunohistochemistry. These results provide transcriptomic evidence that deregulated JNK signaling may impair collagen synthesis in osteoblasts and imply a therapeutic value of JNK inhibitors for treating osteoporosis and preventing skeletal aging by counteracting the age-related reduction in the function of osteoblasts.

Mutations in the insulin (INS) gene may cause permanent neonatal diabetes mellitus (PNDM). Ins2 mutant mouse models provided important insights into the disease mechanisms of PNDM but have limitations for translational research. To establish a large animal model of PNDM, we generated INS(C94Y) transgenic pigs. A line expressing high levels of INS(C94Y) mRNA (70-86% of wild-type INS transcripts) exhibited elevated blood glucose soon after birth but unaltered β-cell mass at the age of 8 days. At 4.5 months, INS(C94Y) transgenic pigs exhibited 41% reduced body weight, 72% decreased β-cell mass (-53% relative to body weight), and 60% lower fasting insulin levels compared with littermate controls. β-cells of INS(C94Y) transgenic pigs showed a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum. Cataract development was already visible in 8-day-old INS(C94Y) transgenic pigs and became more severe with increasing age. Diabetes-associated pathological alterations of kidney and nervous tissue were not detected during the observation period of 1 year. The stable diabetic phenotype and its rescue by insulin treatment make the INS(C94Y) transgenic pig an attractive model for insulin supplementation and islet transplantation trials, and for studying developmental consequences of maternal diabetes mellitus.

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.

Glioblastoma multiforme (GBM) is an aggressive brain tumor with high risks of recurrence and mortality. Chemoradiotherapy resistance has been considered a major factor contributing to the extremely poor prognosis of GBM patients. Therefore, there is an urgent need to develop highly effective therapeutic agents. Here, we demonstrate the anti-tumor effect of morusin, a typical prenylated flavonoid, in GBM through in vivo and in vitro models. Morusin showed selective cytotoxicity toward GBM cell lines without harming normal human astrocytes when the concentration was less than 20 µM. Morusin treatment significantly induced apoptosis of GBM cells, accompanied by the activation of endoplasmic reticulum (ER) stress, and the appearance of cytoplasmic vacuolation and autophagosomes in cells. Then, we found the ER stress activation and cytotoxicity of morusin were rescued by ER stress inhibitor 4-PBA. Furthermore, morusin arrested cell cycle at the G1 phase and inhibited cell proliferation of GBM cells through the Akt-mTOR-p70S6K pathway. Dysregulation of ERs and cell cycle in morusin exposed GBM cells were confirmed by RNA-seq analysis. Finally, we demonstrated the combination of morusin and TMZ remarkably enhanced ER stress and displayed a synergistic effect in GBM cells, and suppressed tumor progression in an orthotopic xenograft model. In conclusion, these findings reveal the toxicity of morusin to GBM cells and its ability to enhance drug sensitivity to TMZ, suggesting the potential application value of morusin in the development of therapeutic strategies for human GBM.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Cell-laden hydrogel microspheres with uniform size show great potential for tissue repair and drug screening applications. Droplet microfluidic systems have been widely used for the generation of cell-laden hydrogel microspheres. However, existing droplet microfluidic systems are mostly based on complex chips and are not compatible with well culture plates. Moreover, microspheres produced by droplet microfluidics need demulsification and purification from oil, which requires time and effort and may compromise cell viability. Herein, we present a simple one-step approach for producing and purifying hydrogel microspheres with an easily assembled microfluidic device. Droplets were generated and solidified in the device tubing. The obtained hydrogel microspheres were then transferred to a tissue culture plate filled with cell culture media and demulsified through evaporation of the oil at 37°C. The removal of oil caused the gelled microspheres to be released into the cell culture media. The encapsulated cells demonstrated good viability and grew into tumor spheroids in 12-14 days. Single cell-laden hydrogel microspheres were also obtained and grown into spheroid in 14 days. This one-step microsphere generation method shows good potential for applications in automated spheroid and organoid cultures as well as drug screening, and could potentially offer benefits for translation of cell/microgel technologies.