Barbara Siegenthaler

A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of β-Amyloid production

Alzheimers disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides, which are generated from amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aβ levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aβ production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled β-secretase endosomal recycling to the plasma membrane and thus affected Aβ production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aβ levels and show how systems biology approaches can unravel the molecular complexity underlying AD.

Retromers in Alzheimer’s Disease

Amyloid-β peptide (Aβ), the key pathogenic agent in Alzheimer’s disease (AD), is released after sequential proteolytic cleavage of the transmembrane amyloid precursor protein (APP). β-Site APP-cleaving enzyme 1 (BACE1) cleaves APP in early endosomes, and the cause of increased BACE cleavage of APP in AD is not fully resolved yet. It has been proposed that perturbed intracellular trafficking of APP, which leads to prolonged residence time in early endosomes, influences Aβ production and hence the risk for AD. Retromers are a family of proteins that mediate the retrieval of transmembrane proteins from the endosomes to the trans-Golgi network. Misregulation of retromers or retromer-associated proteins influences endosomal localization of APP/BACE1. Here we review the role of retromers in the amyloidogenic processing of APP and their pathogenic role in AD.

The epigenetically active small chemical N-methyl pyrrolidone (NMP) prevents estrogen depletion induced osteoporosis

Currently, there are several treatments for osteoporosis however; they all display some sort of limitation and/or side effects making the need for new treatments imperative. We have previously demonstrated that NMP is a bioactive drug which enhances bone regeneration in vivo and acts as an enhancer of bone morphogenetic protein (BMP) in vitro. NMP also inhibits osteoclast differentiation and attenuates bone resorption. In the present study, we tested NMP as a bromodomain inhibitor and for osteoporosis prevention on ovariectomized (OVX) induced rats while treated systemically with NMP. Female Sprague-Dawley rats were ovariectomized and weekly NMP treatment was administrated 1 week after surgery for 15 weeks. Bone parameters and related serum biomarkers were analyzed. 15 weeks of NMP treatment decreased ovariectomy-induced gained weight in average by 43% and improved bone mineral density (BMD) and bone volume over total volume (BV/TV) in rat femur on average by 25% and 41% respectively. Moreover, mineral apposition rate and bone biomarkers of bone turnover in the treatment group were at similar levels with those of the Sham group. Due to the function of NMP as a low affinity bromodomain inhibitor and its mechanism of action involving osteoblasts/osteoclasts balance and inhibitory effect on inflammatory cytokines, NMP is a promising therapeutic compound for the prevention of osteoporosis.

N -methyl pyrrolidone (NMP) inhibits lipopolysaccharide-induced inflammation by suppressing NF-κB signaling

ObjectiveN-methyl pyrrolidone (NMP), a small bioactive molecule, stimulates bone formation and inhibits osteoclast differentiation and bone resorption. The present study was aimed to evaluate the anti-inflammatory potentials of NMP on the inflammatory process and the underlying molecular mechanisms in RAW264.7 macrophages.Materials and methodsRAW264.7 macrophages and mouse primary bone marrow macrophages (mBMMs) were used as an in vitro model to investigate inflammatory processes. Cells were pre-treated with or without NMP and then stimulated with lipopolysaccharides (LPS). The productions of cytokines and NO were determined by proteome profiler method and nitrite analysis, respectively. The expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were measured by Western blotting and/or qPCR. Western blot, ELISA-base reporter assay, and immunofluorescence were used to evaluate the activation of MAP kinases and NF-κB.ResultsLPS-induced mRNA expressions of TNF-α, IL-1β, IL-6, iNOS, and COX-2 were inhibited by NMP in a dose-dependent manner. NMP also suppressed the LPS-increased productions of iNOS and NO. The proteome profiler array showed that several cytokines and chemokines involved in inflammation and up-regulated by LPS stimulation were significantly down-regulated by NMP. Additionally, this study shows that the effect of NMP is mediated through down-regulation of NFκB pathway.ConclusionsOur results show that NMP inhibits the inflammatory mediators in macrophages by an NFκB-dependent mechanism, based on the epigenetical activity of NMP as bromodomain inhibitor. In the light of its action on osteoblast and osteoclast differentiation process and its anti-inflammatory potential, NMP might be used in inflammation-related bone loss.

Effects of Stem Cell Factor on Cell Homing During Functional Pulp Regeneration in Human Immature Teeth.

Conventional root canal treatment in immature permanent teeth can lead to early tooth loss in children because root formation is discontinued. We investigated whether the stem cell factor (SCF) could facilitate cell homing in the pulpless immature root canal and promote regeneration of a functional pulp. In vitro, human mesenchymal stem cells (hMSCs) were exposed to SCF at various concentrations for assessing cell migration, proliferation, and differentiation toward odonto/osteoblasts by 3D-chemotaxis slides, WST-1 assay, and alkaline phosphatase activity, respectively. Fibrin gels were used to deliver 15 μg/mL SCF for in vivo experiments. The release kinetic of SCF was assessed in vitro. Two corresponding human immature premolars, with or without SCF, were placed at rat calvariae for 6 and 12 weeks. All tooth specimens were either analyzed histologically and the percentage of tissue ingrowth determined or the cells were extracted from the pulp space, and the mRNA level of DMP1, DSPP, Col1, NGF, and VEGF were assessed by quantitative polymerase chain reaction. In the presence of SCF, we saw an increase in hMSCs directional migration, proliferation, and odonto/osteogenic differentiation. SCF also increased the extent of tissue ingrowth at 6 weeks but not at 12 weeks. However, at this time point, the formed tissue appeared more mature in samples with SCF. In terms of gene transcription, DMP1, Col1, and VEGF were the significantly upregulated genes, while DSPP and NGF were not affected. Our results suggest that SCF can accelerate cell homing and the maturation of the pulp-dentin complex in human immature teeth.

Comparative study of NMP-preloaded and dip-loaded membranes for guided bone regeneration of rabbit cranial defects

Guided bone regeneration (GBR) has been utilized for several decades for the healing of cranio‐maxillofacial bone defects and, particularly in the dental field, by creating space with a barrier membrane to exclude soft tissue and encourage bone growth in the membrane‐protected volume. Although the first membranes were non‐resorbable, a new generation of GBR membranes aims to biodegrade and provide bioactivity for better overall results. The Inion GTR™ poly(lactide‐co‐glycolide) (PLGA) membrane is not only resorbable but also bioactive, since it includes N‐methylpyrrolidone (NMP), which has been shown to promote bone regeneration. In this study, the effects of loading different amounts of NMP onto the membrane through chemical vapour deposition or dipping have been explored. In vitro release demonstrated that lower levels of NMP led to lower NMP concentrations and slower release, based on total NMP loaded in the membrane. The dipped membrane released almost all of the NMP within 15 min, leading to a high NMP concentration. For the in vivo studies in rabbits, 6 mm calvarial defects were created and left untreated or covered with an ePTFE membrane or PLGA membranes dipped in, or preloaded with, NMP. Evaluation of the bony regeneration revealed that the barrier membranes improved bony healing and that a decrease in NMP content improved the performance. Overall, we have demonstrated the potential of these PLGA membranes with a more favourable NMP release profile and the significance of exploring the effect of NMP on these PLGA membranes with regard to bone ingrowth. Copyright

Fibrin Hydrogel Based Bone Substitute Tethered with BMP-2 and BMP-2/7 Heterodimers

Current clinically used delivery methods for bone morphogenetic proteins (BMPs) are collagen based and require large concentrations that can lead to dangerous side effects. Fibrin hydrogels can serve as osteoinductive bone substitute materials in non-load bearing bone defects in combination with BMPs. Two strategies to even further optimize such a fibrin based system include employing more potent BMP heterodimers and engineering growth factors that can be covalently tethered to and slowly released from a fibrin matrix. Here we present an engineered BMP-2/BMP-7 heterodimer where an N-terminal transglutaminase substrate domain in the BMP-2 portion provides covalent attachment to fibrin together with a central plasmin substrate domain, a cleavage site for local release of the attached BMP-2/BMP-7 heterodimer under the influence of cell-activated plasmin. In vitro and in vivo results revealed that the engineered BMP-2/BMP-7 heterodimer induces significantly more alkaline phosphatase activity in pluripotent cells and bone formation in a rat calvarial model than the engineered BMP-2 homodimer. Therefore, the engineered BMP-2/BMP-7 heterodimer could be used to reduce the amount of BMP needed for clinical effect.

The Bromodomain Inhibitor N-Methyl pyrrolidone Prevents Osteoporosis and BMP-Triggered Sclerostin Expression in Osteocytes

(1) Background: In an adult skeleton, bone is constantly renewed in a cycle of bone resorption, followed by bone formation. This coupling process, called bone remodeling, adjusts the quality and quantity of bone to the local needs. It is generally accepted that osteoporosis develops when bone resorption surpasses bone formation. Osteoclasts and osteoblasts, bone resorbing and bone forming cells respectively, are the major target in osteoporosis treatment. Inside bone and forming a complex network, the third and most abundant cells, the osteocytes, have long remained a mystery. Osteocytes are responsible for mechano-sensation and -transduction. Increased expression of the osteocyte-derived bone inhibitor sclerostin has been linked to estrogen deficiency-induced osteoporosis and is therefore a promising target for osteoporosis management. (2) Methods: Recently we showed in vitro and in vivo that NMP (N-Methyl-2-pyrrolidone) is a bioactive drug enhancing the BMP-2 (Bone Morphogenetic Protein 2) induced effect on bone formation while blocking bone resorption. Here we tested the effect of NMP on the expression of osteocyte-derived sclerostin. (3) Results: We found that NMP significantly decreased sclerostin mRNA and protein levels. In an animal model of osteoporosis, NMP prevented the estrogen deficiency-induced increased expression of sclerostin. (4) Conclusions: These results support the potential of NMP as a novel therapeutic compound for osteoporosis management, since it preserves bone by a direct interference with osteoblasts and osteoclasts and an indirect one via a decrease in sclerostin expression by osteocytes.

N,N Dimethylacetamide a drug excipient that acts as bromodomain ligand for osteoporosis treatment

N,N-Dimethylacetamide (DMA) is a water-miscible solvent, FDA approved as excipient and therefore widely used as drug-delivery vehicle. As such, DMA should be devoid of any bioactivity. Here we report that DMA is epigenetically active since it binds bromodomains and inhibits osteoclastogenesis and inflammation. Moreover, DMA enhances bone regeneration in vivo. Therefore, our in vivo and in vitro data reveal DMA’s potential as an anti-osteoporotic agent via the inhibition of osteoclast mediated bone resorption and enhanced bone regeneration. Our results highlight the potential therapeutic benefits of DMA and the need for reconsideration of previous reports where DMA was used as an ‘inactive’ drug-delivery vehicle.