Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells.

Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

It takes two to tango: cardiac fibroblast-derived NO-induced cGMP enters cardiac myocytes and increases cAMP by inhibiting PDE3.

The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing ß-receptor-induced contractile responses appears as one of NO/cGMP's functions in the non-failing heart.

Mutation of Tyrosine Sites in the Human Alpha-Synuclein Gene Induces Neurotoxicity in Transgenic Mice with Soluble Alpha-Synuclein Oligomer Formation.

Overexpression of α-synuclein with tyrosine mutated to phenylalanine at position 125 leads to a severe phenotype with motor impairment and neuropathology in Drosophila. Here, we hypothesized that tyrosine mutations would similarly lead to impaired motor performance with neuropathology in a rodent model. In transgenic mice (ASO), tyrosines at positions 125, 133, and 136 in human α-synuclein were mutated to phenylalanine and cloned into a Thy1.2 expression vector, which was used to create transgenic mouse lines on a mixed genetic background TgN(Thy-1-SNCA-YF)4Emfu (YF). The YF mice had a decreased lifespan and displayed a dramatic motor phenotype with paralysis of both hind- and forelegs. Post-translational modification of α-synuclein due to phosphorylation of serine 129 is often seen in inclusions in the brains of patients with α-synucleinopathies. We observed a slight but significant increase in phosphorylation of serine 129 in the cytosol in YF mice compared to age-matched human α-synuclein transgenic mice (ASO). Conversely, significantly decreased phosphorylation of serine 129 was seen in synaptosomes of YF mice that also contained higher amounts of soluble oligomers. YF mice deposited full-length α-synuclein aggregates in neurons widespread in the CNS with the main occurrence in the forebrain structures of the cerebral cortex, the basal ganglia, and limbic structures. Full-length α-synuclein labeling was also prominent in many nuclear regions of the brain stem, deep cerebellar nuclei, and cerebellar cortex. The study shows that the substitution of tyrosines to phenylalanine in α-synuclein at positions 125, 133, and 136 leads to severe toxicity in vivo. An insignificant change upon tyrosine substitution suggests that the phosphorylation of serine 129 is not the cause of the toxicity.

Expression of the miR-302/367 microRNA cluster is regulated by a conserved long non-coding host-gene.

MicroRNAs are important regulators of cellular functions. MiR-302/367 is a polycistronic miRNA cluster that can induce and maintain pluripotency. Here we investigate the transcriptional control and the processing of the miR-302 host-gene in mice. Our results indicate that the mmu-miR-302 host-gene is alternatively spliced, polyadenylated and exported from the nucleus. The regulatory sequences extend at least 2 kb upstream of the transcription start site and contain several conserved binding sites for both transcriptional activators and repressors. The gene structure and regulatory elements are highly conserved between mouse and human. So far, regulating miR-302 expression is the only known function of the miR-302 host-gene. Even though we here only provide one example, regulation of microRNA transcription might be a so far little recognized function of long non-coding RNA genes.

AMPA Induces NO-Dependent cGMP Signals in Hippocampal and Cortical Neurons via L-Type Voltage-Gated Calcium Channels.

The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a "knock-in" mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100 nM and 3 µM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.

Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts.

BACKGROUND AND PURPOSE: The intracellular signalling molecule cGMP, formed by NO-sensitive GC (NO-GC), has an established function in the vascular system. Despite numerous reports about NO-induced cGMP effects in the heart, the underlying cGMP signals are poorly characterized. EXPERIMENTAL APPROACH: Therefore, we analysed cGMP signals in cardiac myocytes and fibroblasts isolated from knock-in mice expressing a FRET-based cGMP indicator. KEY RESULTS: Whereas in cardiac myocytes, none of the known NO-GC-activating substances (NO, GC activators, and GC stimulators) increased cGMP even in the presence of PDE inhibitors, they induced substantial cGMP increases in cardiac fibroblasts. As cardiac myocytes and fibroblasts are electrically connected via gap junctions, we asked whether cGMP can take the same route. Indeed, in cardiomyocytes co-cultured on cardiac fibroblasts, NO-induced cGMP signals were detectable, and two groups of unrelated gap junction inhibitors abolished these signals. CONCLUSION AND IMPLICATION: We conclude that NO-induced cGMP formed in cardiac fibroblasts enters cardiac myocytes via gap junctions thereby turning cGMP into an intercellular signalling molecule. The findings shed new light on NO/cGMP signalling in the heart and will potentially broaden therapeutic opportunities for cardiac disease.

Isolation of cancer stem cells by selection for miR-302 expressing cells.

BACKGROUND: Cancer stem cells are believed to be a major reason for long-term therapy failure because they are multi-drug resistant and able to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more 'differentiated', i.e., less stem cell-like tumor cells. METHODS: Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. A selectable egfp-neo coding sequence was inserted in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start site was used. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. RESULTS: After three weeks, tumors were removed for analysis and primary cultures were established. Stem cell-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it was not the original ES cells that had been isolated. CONCLUSIONS: We show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells.

The choroid plexus sodium-bicarbonate cotransporter NBCe2 regulates mouse cerebrospinal fluid pH.

KEY POINTS: Normal pH is crucial for proper functioning of the brain, and disorders increasing the level of CO2 in the blood lead to a decrease in brain pH. CO2 can easily cross the barriers of the brain and will activate chemoreceptors leading to an increased exhalation of CO2 . The low pH, however, is harmful and bases such as HCO3- are imported across the brain barriers in order to normalize brain pH. We show that the HCO3- transporter NBCe2 in the choroid plexus of the blood-cerebrospinal fluid barrier is absolutely necessary for normalizing CSF pH during high levels of CO2 . This discovery represents a significant step in understanding the molecular mechanisms behind regulation of CSF pH during acid-base disturbances, such as chronic lung disease. ABSTRACT: The choroid plexus epithelium (CPE) is located in the brain ventricles where it produces the majority of the cerebrospinal fluid (CSF). The hypothesis that normal brain function is sustained by CPE-mediated CSF pH regulation by extrusion of acid-base equivalents was tested by determining the contribution of the electrogenic Na+ -HCO3- cotransporter NBCe2 to CSF pH regulation. A novel strain of NBCe2 (Slc4a5) knockout (KO) mice was generated and validated. The base extrusion rate after intracellular alkalization was reduced by 77% in NBCe2 KO mouse CPE cells compared to control mice. NBCe2 KO mice and mice with CPE-targeted NBCe2 siRNA knockdown displayed a reduction in CSF pH recovery during hypercapnia-induced acidosis of approximately 85% and 90%, respectively, compared to control mice. NBCe2 KO did not affect baseline respiration rate or tidal volume, and the NBCe2 KO and wild-type (WT) mice displayed similar ventilatory responses to 5% CO2 exposure. NBCe2 KO mice were not protected against pharmacological or heating-induced seizure development. In conclusion, we establish the concept that the CPE is involved in the regulation of CSF pH by demonstrating that NBCe2 is necessary for proper CSF pH recovery after hypercapnia-induced acidosis.

Plekhg5-regulated autophagy of synaptic vesicles reveals a pathogenic mechanism in motoneuron disease.

Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures. Plekhg5 gene inactivation in mice results in a late-onset motoneuron disease, characterized by degeneration of axon terminals. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26. These findings define a mechanism for regulating autophagy in neurons that specifically targets synaptic vesicles. Disruption of this mechanism may contribute to the pathophysiology of several forms of motoneuron disease.

Constitutive transgene expression of Stem Cell Antigen-1 in the hair follicle alters the sensitivity to tumor formation and progression.

The cell surface protein Stem Cell Antigen-1 (Sca-1) marks stem or progenitor cells in several murine tissues and is normally upregulated during cancer development. Although the specific function of Sca-1 remains unknown, Sca-1 seems to play a role in proliferation, differentiation and cell migration in a number of tissues. In the skin epithelium, Sca-1 is highly expressed in the interfollicular epidermis but is absent in most compartments of the hair follicle; however, the function of Sca-1 in the skin has not been investigated. To explore the role of Sca-1 in normal and malignant skin development we generated transgenic mice that express Sca-1 in the hair follicle stem cells that are normally Sca-1 negative. Development of hair follicles and interfollicular epidermis appeared normal in Sca-1 mutant mice; however, follicular induction of Sca-1 expression in bulge region and isthmus stem cells reduced the overall yield of papillomas in a chemical carcinogenesis protocol. Despite that fewer papillomas developed in transgenic mice a higher proportion of the papillomas underwent malignant conversion. These findings suggest that overexpression of Sca-1 in the hair follicle stem cells contributes at different stages of tumour development. In early stages, overexpression of Sca-1 decreases tumour formation while at later stages overexpression of Sca-1 seems to drive tumours towards malignant progression.

Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System.

Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively.

C4.4A gene ablation is compatible with normal epidermal development and causes modest overt phenotypes.

C4.4A is a modular glycolipid-anchored Ly6/uPAR/α-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.

Distinct Functions for Anterograde and Retrograde Sorting of SORLA in Amyloidogenic Processes in the Brain.

SORLA is a neuronal sorting receptor implicated both in sporadic and familial forms of AD. SORLA reduces the amyloidogenic burden by two mechanisms, either by rerouting internalized APP molecules from endosomes to the trans-Golgi network (TGN) to prevent proteolytic processing or by directing newly produced Aß to lysosomes for catabolism. Studies in cell lines suggested that the interaction of SORLA with cytosolic adaptors retromer and GGA is required for receptor sorting to and from the TGN. However, the relevance of anterograde or retrograde trafficking for SORLA activity in vivo remained largely unexplored. Here, we generated mouse models expressing SORLA variants lacking binding sites for GGA or retromer to query this concept in the brain. Disruption of retromer binding resulted in a retrograde-sorting defect with accumulation of SORLA in endosomes and depletion from the TGN, and in an overall enhanced APP processing. In contrast, disruption of the GGA interaction did not impact APP processing but caused increased brain Aß levels, a mechanism attributed to a defect in anterograde lysosomal targeting of Aß. Our findings substantiated the significance of adaptor-mediated sorting for SORLA activities in vivo, and they uncovered that anterograde and retrograde sorting paths may serve discrete receptor functions in amyloidogenic processes. SIGNIFICANCE STATEMENT: SORLA is a sorting receptor that directs target proteins to distinct intracellular compartments in neurons. SORLA has been identified as a genetic risk factor for sporadic, but recently also for familial forms of AD. To confirm the relevance of SORLA sorting for AD processes in the brain, we generated mouse lines, which express trafficking mutants instead of the wild-type form of this receptor. Studying neuronal activities in these mutant mice, we dissected distinct trafficking routes for SORLA guided by two cytosolic adaptors termed GGA and retromer. We show that these sorting pathways serve discrete functions in control of amyloidogenic processes and may represent unique therapeutic targets to interfere with specific aspects of neurodegenerative processes in the diseased brain.

The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses.

Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.

Lysosomal sorting of amyloid-ß by the SORLA receptor is impaired by a familial Alzheimer's disease mutation.

SORLA/SORL1 is a unique neuronal sorting receptor for the amyloid precursor protein that has been causally implicated in both sporadic and autosomal dominant familial forms of Alzheimer's disease (AD). Brain concentrations of SORLA are inversely correlated with amyloid-ß (Aß) in mouse models and AD patients, suggesting that increasing expression of this receptor could be a therapeutic option for decreasing the amount of amyloidogenic products in affected individuals. We characterize a new mouse model in which SORLA is overexpressed, and show a decrease in Aß concentrations in mouse brain. We trace the underlying molecular mechanism to the ability of this receptor to direct lysosomal targeting of nascent Aß peptides. Aß binds to the amino-terminal VPS10P domain of SORLA, and this binding is impaired by a familial AD mutation in SORL1. Thus, loss of SORLA's Aß sorting function is a potential cause of AD in patients, and SORLA may be a new therapeutic target for AD drug development.

SORLA-dependent and -independent functions for PACS1 in control of amyloidogenic processes.

Sorting-related receptor with A-type repeats (SORLA) is a sorting receptor for the amyloid precursor protein (APP) that prevents breakdown of APP into Aß peptides, a hallmark of Alzheimer's disease (AD). Several cytosolic adaptors have been shown to interact with the cytoplasmic domain of SORLA, thereby controlling intracellular routing of SORLA/APP complexes in cell lines. However, the relevance of adaptor-mediated sorting of SORLA for amyloidogenic processes in vivo remained unexplored. We focused on the interaction of SORLA with phosphofurin acidic cluster sorting protein 1 (PACS1), an adaptor that shuttles proteins between the trans-Golgi network (TGN) and endosomes. By studying PACS1 knockdown in neuronal cell lines and investigating transgenic mice expressing a PACS1-binding-defective mutant form of SORLA, we found that disruption of SORLA and PACS1 interaction results in the inability of SORLA/APP complexes to sort to the TGN in neurons and in increased APP processing in the brain. Loss of PACS1 also impairs the proper expression of the cation-independent mannose 6-phosphate receptor and its target cathepsin B, a protease that breaks down Aß. Thus, our data identified the importance of PACS1-dependent protein sorting for amyloidogenic-burden control via both SORLA-dependent and SORLA-independent mechanisms.

The proto-oncogene TWIST1 is regulated by microRNAs.

Upregulation of the proto-oncogene Twist1 is highly correlated with acquired drug resistance and poor prognosis in human cancers. Altered expression of this multifunctional transcription factor is also associated with inherited skeletal malformations. The mammalian Twist1 3'UTRs are highly conserved and contain a number of potential regulatory elements including miRNA target sites. We analyzed the translational regulation of TWIST1 using luciferase reporter assays in a variety of cell lines. Among several miRNAs tested, miR-145a-5p, miR-151-5p and a combination of miR-145a-5p + miR-151-5p and miR-151-5p + miR-337-3p were able to significantly repress Twist1 translation. This phenomena was confirmed with both exogenous and endogenous miRNAs and was dependent on the presence of the predicted target sites in the 3'UTR. Furthermore, the repression was sensitive to LNA-modified miRNA antagonists and resulted in decreased migratory potential of murine embryonic fibroblast cells. Understanding the in vivo mechanisms of this oncogene's regulation might open up a possibility for therapeutic interference by gene specific cancer therapies.

Septin9 is involved in T-cell development and CD8+ T-cell homeostasis.

SEPTIN9 (SEPT9) is a filament-forming protein involved in numerous cellular processes. We have used a conditional knock out allele of Sept9 to specifically delete Sept9 in T-cells. As shown by fluorescence-activated cell sorting, loss of Sept9 at an early thymocyte stage in the thymus results in increased numbers of double-negative cells indicating that SEPT9 is involved in the transition from the double-negative stage during T-cell development. Accordingly, the relative numbers of mature T-cells in the periphery are decreased in mice with a T-cell-specific deletion of Sept9. Proliferation of Sept9-deleted CD8(+) T-cells from the spleen is decreased upon stimulation in culture. The altered T-cell homeostasis caused by the loss of Sept9 results in an increase of CD8(+) central memory T-cells.