Marcus Niebert

Subcellular compartment targeting of layered double hydroxide nanoparticles

Current investigations show that layered double hydroxide (LDH) nanoparticles have high potential as effective non-viral agents for cellular drug delivery due to their low cytotoxicity, good biocompatibility, high drug loading, control of particle size and shape, targeted delivery and drug release control. Two types of Mg(2)Al-LDH nanoparticles with fluorescein isothiocyanate (FITC) were controllably prepared. One is morphologically featured as typical hexagonal sheets (50-150 nm laterally wide and 10-20 nm thick), while the other as typical rods (30-60 nm wide and 100-200 nm long). These LDH(FTIC) nanoparticles are observed to immediately transfect into different mammalian cell lines. We found that internalized LDH(FITC) nanorods are quickly translocated into the nucleus while internalized LDH(FITC) nanosheets are retained in the cytoplasm. Inhibition experiments show that the cellular uptake is a clathrin-mediated time- and concentration-dependent endocytosis. Endosomal escape of LDH(FITC) nanoparticles is suggested to occur through the deacidification of LDH nanoparticles. Since quick nuclear targeting of LDH(FITC) nanorods requires an active process, and although the exact mechanism is yet to be fully understood, it probably involves an active transport via microtubule-mediated trafficking processes. Targeted addressing of two major subcellular compartments by simply controlling the particle morphology/size could find a number of applications in cellular biomedicine.

Efficient siRNA delivery to mammalian cells using layered double hydroxide nanoparticles

Although siRNAs have surpassed expectations in experiments to alter gene expression in vitro, the lack of an efficient in vivo delivery system still remains a challenge in siRNA therapeutics development and has been recognized as a major hurdle for clinical applications. In this paper we describe an inorganic nanoparticle-based delivery system that is readily adaptable for in vivo systems. Layered double hydroxide (LDH) nanoparticles, a family of inorganic crystals, tightly bind, protect, and release siRNA molecules and deliver them efficiently to mammalian cells in vitro. The uptake of siRNA-loaded LDH nanoparticles occurs via endocytosis, whereby the nanoparticles dissolve due to the low pH in the endosome, thereby aiding endosomal escape into the cytoplasm. The influence of LDH nanoparticles on cell viability and proliferation is negligible at concentrations <or=0.050 mg mL(-1), and a pronounced down-regulation of protein expression upon LDH mediated siRNA transfection of HEK293T cells is observed.

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking

Serotonin receptors 5-HT1A and 5-HT7 are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT1A and 5-HT7 receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT1A or 5-HT7 receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT7–5-HT7 homodimers, followed by the 5-HT7–5-HT1A heterodimers and 5-HT1A–5-HT1A homodimers. Functionally, heterodimerization decreases 5-HT1A-receptor-mediated activation of Gi protein without affecting 5-HT7-receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT1A receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT1A receptor internalization and also enhances the ability of the 5-HT1A receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT7 receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT1A–5-HT7 heterodimers and, consequently, their functional importance undergoes pronounced developmental changes.

Serotonin receptor 1A–modulated phosphorylation of glycine receptor α3 controls breathing in mice

Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A-specific (5-HTR1A-specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A-GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A-GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction.

An Ion-insensitive cAMP Biosensor for Long Term Quantitative Ratiometric Fluorescence Resonance Energy Transfer (FRET) Measurements under Variable Physiological Conditions

Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT1A receptor.

Expression and Function of Serotonin 2A and 2B Receptors in the Mammalian Respiratory Network

Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs) that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT2A, 5-HT2B, and 5-HT2C receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT2ARs, expression and function of 5-HT2BRs within the respiratory network are still unclear. 5-HT2BR utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT2ARs and 5-HT2BRs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT2BR. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation.

Intercellular Transport of Oct4 in Mammalian Cells: A Basic Principle to Expand a Stem Cell Niche?

Background The octamer-binding transcription factor 4 (Oct4) was originally described as a marker of embryonic stem cells. Recently, the role of Oct4 as a key regulator in pluripotency was shown by its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. While artificial induction of pluripotency using transcription factors is possible in mammalian cell culture, it remains unknown whether a potential natural transfer mechanism might be of functional relevance in vivo. The stem cell based regeneration of deer antlers is a unique model for rapid and complete tissue regeneration in mammals and therefore most suitable to study such mechanisms. Here, the transfer of pluripotency factors from resident stem cell niche cells to differentiated cells could recruit more stem cells and start rapid tissue regeneration. Methodology/Principal Findings We report on the ability of STRO-1+ deer antlerogenic mesenchymal stem cells (DaMSCs) to transport Oct4 via direct cell-to-cell connections. Upon cultivation in stem cell expansion medium, we observed nuclear Oct4 expression in nearly all cells. A number of these cells exhibit Oct4 expression not only in the nucleus, but also with perinuclear localisation and within far-ranging intercellular connections. Furthermore, many cells showed intercellular connections containing both F-actin and α-tubulin and through which transport could be observed. To proof that intercellular Oct4-transfer has functional consequences in recipient cells we used a co-culture approach with STRO-1+ DaMSCs and a murine embryonic fibroblast indicator cell line (Oct4-GFP MEF). In this cell line a reporter gene (GFP) under the control of an Oct4 responsive element is only expressed in the presence of Oct4. GFP expression in Oct4-GFP cells started after 24 hours of co-culture providing evidence of Oct4 transfer from STRO-1+ DaMSCs to Oct4-GFP MEF target cells. Conclusions Our findings indicate a possible mechanism for the expansion of a resident stem cell niche by induction of pluripotency in surrounding non-niche cells via transfer of transcription factors through intercellular connections. This provides a new approach to explain the rapid annual antler regrowth.

Rescue of Cyclic AMP Mediated Long Term Potentiation Impairment in the Hippocampus of Mecp2 Knockout (Mecp2-/y) Mice by Rolipram

Rett syndrome (RTT) patients experience learning difficulties and memory loss. Analogous deficits of hippocampal plasticity are reported in mouse models of RTT. To elucidate the underlying pathophysiology, we studied long term potentiation (LTP) at the CA3 to CA1 synapses in the hippocampus in acute brain slices from WT and Mecp2-/y mice, by either activating cAMP dependent pathway or using high frequency stimulation, by means of patch clamp. We have observed that, the NMDA channel current characteristics remain unchanged in the Mecp2-/y mice. The adenylyl cyclase (AC) agonist forskolin evoked a long lasting potentiation of evoked EPSCs in WT CA1 neurons, but only minimally enhanced the EPSCs in the Mecp2-/y mice. This weaker potentiation in Mecp2-/y mice was ameliorated by application of phosphodiesterase 4 inhibitor rolipram. The hyperpolarization activated cyclic nucleotide gated channel current (Ih) was potentiated to similar extent by forskolin in both phenotypes. Multiple tetanus induced cAMP-dependent plasticity was also impaired in the Mecp2-/y mice, and was also partially rescued by rolipram. Western blot analysis of CA region of Mecp2-/y mice hippocampus revealed more than twofold up-regulation of protein kinase A (PKA) regulatory subunits, while the expression of the catalytic subunit remained unchanged. We hypothesize that the overexpressed PKA regulatory subunits buffer cAMP and restrict the PKA mediated phosphorylation of target proteins necessary for LTP. Blocking the degradation of cAMP, thereby saturating the regulatory subunits alleviated this defect.

Analysis of the serotonergic system in a mouse model of Rett syndrome reveals unusual upregulation of serotonin receptor 5b

Mutations in the transcription factor methyl-CpG-binding-protein 2 (MeCP2) cause a delayed-onset neurodevelopmental disorder known as Rett syndrome (RTT). Although alteration in serotonin levels have been reported in RTT patients, the molecular mechanisms underlying these defects are not well understood. Therefore, we chose to investigate the serotonergic system in hippocampus and brainstem of male Mecp2-/y knock-out mice in the B6.129P2(C)-Mecp2(tm1.1Bird) mouse model of RTT. The serotonergic system in mouse is comprised of 16 genes, whose mRNA expression profile was analyzed by quantitative RT-PCR. Mecp2-/y mice are an established animal model for RTT displaying most of the cognitive and physical impairments of human patients and the selected areas receive significant modulation through serotonin. Using anatomically and functional characterized areas, we found region-specific differential expression between wild type and Mecp2-/y mice at post-natal day 40. In brainstem, we found five genes to be dysregulated, while in hippocampus, two genes were dysregulated. The one gene dysregulated in both brain regions was dopamine decarboxylase, but of special interest is the serotonin receptor 5b (5-ht5b), which showed 75-fold dysregulation in brainstem of Mecp2-/y mice. This dysregulation was not due to upregulation, but due to failure of down-regulation in Mecp2-/y mice during development. Detailed analysis of 5-ht5b revealed a receptor that localizes to endosomes and interacts with Gαi proteins.

GlyT2-Dependent Preservation of MECP2-Expression in Inhibitory Neurons Improves Early Respiratory Symptoms but Does Not Rescue Survival in a Mouse Model of Rett Syndrome

Mutations in methyl-CpG-binding protein 2 (MECP2) gene have been shown to manifest in a neurodevelopmental disorder that is called Rett syndrome. A typical problem that occurs during development is a disturbance of breathing. To address the role of inhibitory neurons, we generated a mouse line that restores MECP2 in inhibitory neurons in the brainstem by crossbreeding a mouse line that expresses the Cre-recombinase (Cre) in inhibitory neurons under the control of the glycine transporter 2 (GlyT2, slc6a5) promotor (GlyT2-Cre) with a mouse line that has a floxed-stop mutation of the Mecp2 gene (Mecp2stop/y). Unrestrained whole-body-plethysmography at postnatal day P60 revealed a low respiratory rate and prolonged respiratory pauses in Mecp2stop/y mice. In contrast, GlyT2-Cre positive Mecp2stop/y mice (Cre+; Mecp2stop/y) showed greatly improved respiration and were indistinguishable from wild type littermates. These data support the concept that alterations in inhibitory neurons are important for the development of the respiratory phenotype in Rett syndrome.