Podcasts about Catenin

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548410v1?rss=1 Authors: Biswas, S., Shahriar, S., Bachay, G., Arvanitis, P., Brunken, W. J., Agalliu, D. Abstract: Interactions among neuronal, glial and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Although synaptic dysfunctions precede vascular abnormalities in many retinal pathologies, how neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single cell RNA sequencing and functional validation, we show that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1-/- retinas, where neurons fail to release glutamate. In contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1-/- retinas, where constitutively depolarized rods release excessive glutamate. Norrin mRNA expression and endothelial Norrin/{beta}-catenin activity are downregulated in Vglut1-/- retinas, and upregulated in Gnat1-/- retinas. Pharmacological activation of endothelial Norrin/{beta}-catenin signaling in Vglut1-/- retinas rescued defects in deep plexus angiogenesis and paracellular BRB integrity. Our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating Norrin/{beta}-catenin signaling. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.01.535227v1?rss=1 Authors: Yamamoto, M., Miyoshi, M., Morioka, K., Mitani, T., Takaya, T. Abstract: A myogenetic oligodeoxynucleotide, iSN04, is the 18-base single-stranded DNA that acts as an anti-nucleolin aptamer. iSN04 has been reported to restore myogenic differentiation by suppressing inflammatory responses in myoblasts isolated from patients with diabetes or healthy myoblasts exposed to cancer-releasing factors. Thus, iSN04 is expected to be a nucleic acid drug for the muscle wasting associated with chronic diseases. The present study investigated the anti-inflammatory mechanism of iSN04 in the murine myoblast cell line C2C12. Tumor necrosis factor- (TNF-) or Toll-like receptor (TLR) ligands (Pam3CSK4 and FSL-1) induced nuclear translocation and transcriptional activity of nuclear factor-{kappa}B (NF-{kappa}B), resulting in upregulated expression of TNF- and interleukin-6. Pre-treatment with iSN04 significantly suppressed these inflammatory responses by inhibiting the nuclear accumulation of {beta}-catenin induced by TNF- or TLR ligands. These results demonstrate that antagonizing nucleolin with iSN04 downregulates the inflammatory effect mediated by the {beta}-catenin/NF-{kappa}B signaling pathway in myoblasts. In addition, the anti-inflammatory effects of iSN04 were also observed in smooth muscle cells and pre-adipocytes, suggesting that iSN04 may be useful in preventing inflammation induced by metabolic disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.21.529411v1?rss=1 Authors: Sun, H., Shami Shah, A., Bonfini, A., Buchon, N., Baskin, J. M. Abstract: The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/{beta}-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, we examine the cellular determinants of ISC proliferation, harnessing kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. We find that Wnt signaling within Prospero-positive cells supports ISC proliferation and that kramer regulates Wnt signaling in this context by antagonizing kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes kramer as a physiological regulator of Wnt/{beta}-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/{beta}-catenin signaling. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.15.528619v1?rss=1 Authors: Abedrabbo, M., Sloomi, S., Abu-leil, R., Cohen-Kfir, E., Ravid, S. Abstract: E-cadherin, - and {beta}-catenin (E-cadherin-catenin complex) together with the cytoskeleton build the core of Adherens junctions (AJs). Scribble and Lgl1 are tumor suppressors, and it has been reported that Scribble stabilizes the coupling of E-cadherin with catenins promoting epithelial cell adhesion, but the molecular mechanism remains unknown. Here, we investigated the role of Scribble, Lgl1, and myosin-IIA (NMII-A) in AJ integrity. We show that Scribble, Lgl1, and NMII-A reside in a complex with the E-cadherin-catenin complex. Depletion of either Scribble or Lgl1 disrupts the localization of E-cadherin-catenin complex to AJs. aPKC{zeta} phosphorylation of Lgl1 regulates AJ localization of Lgl1 and E-cadherin-catenin complex. Both Scribble and Lgl1 regulate the activation and recruitment of NMII-A at AJs. Finally, Scribble and Lgl1 are downregulated by TGF{beta}-induced EMT, and re-expression of Scribble or Lgl1 during EMT impedes its progression. Our results provide insight into the mechanism regulating AJ integrity by Scribble, Lgl1, and NMII-A. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.01.526618v1?rss=1 Authors: Wood, M. N., Wang, Y., Flozak, A. S., Yemelyanov, A., Gottardi, C. J. Abstract: alpha-catenin (alpha-cat) displays force-dependent binding to actin filaments, but the features of adherens junction structure and function most vulnerable to loss of this allosteric mechanism remain to be generalized across epithelial cell-types. By reconstituting an alpha-cat F-actin-binding domain unfolding mutant known to exhibit enhanced binding to actin (alpha-cat-H0-FABD+) into alpha-cat knock-out Madin Darby Canine Kidney (MDCK) cells, we show that partial loss of the alpha-cat catch bond mechanism (via an altered H0 alpha-helix) leads to stronger epithelial sheet integrity with greater co-localization between the alpha-cat-H0-FABD+ mutant and actin. alpha-cat-H0-FABD+ -expressing cells are less efficient at closing scratch-wounds or uniformly packing, suggesting reduced capacity for more dynamic cell-cell coordination. Evidence alpha-cat-H0-FABD+ is equally accessible to the conformationally sensitive alpha18 antibody epitope as WT alpha-cat suggests this mutant favors binding to lower tension cortical actin networks. These data suggest signals that reduce the force-sensitivity of the alpha-cat-cortical actin interaction might improve epithelial monolayer strength through enhanced coupling to lower tension cortical actin networks, but that such association would render alpha-cat less selective of higher-tension actin networks, extracting a cost for dynamic processes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.11.523290v1?rss=1 Authors: Mazzarino, R. C., Perez-Corredor, P. A., Vanderleest, T. E., Vacano, G. N., Sanchez, J., Villalba-Moreno, N. D., Krasemann, S., Mendivil Perez, M. A., Aguillon, D., Jimenez-Del-Rio, M., Baena, A., Sepulveda-Falla, D., Lopera-Restrepo, F., Quiroz-Gaviria, Y., Arboleda-Velasquez, J. F. Abstract: Alzheimer's disease (AD) is the most common cause of dementia among older adults. APOE3 Christchurch (R136S, APOE3Ch) variant homozygosity was reported in an individual with extreme resistance to autosomal dominant AD due to the PSEN1 E280A mutation. This subject had a delayed clinical age at onset and resistance to tauopathy and neurodegeneration despite extremely high amyloid plaque burden. We established induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and from a non-protected kindred control (with PSEN1 E280A and APOE3/3). We used CRISPR/Cas9 gene editing to successfully remove the APOE3Ch to wild type in iPS cells from the protected case and to introduce the APOE3Ch as homozygote in iPS cells from the non-protected case to examine causality. We found significant reduction of tau phosphorylation (pTau 202/205 and pTau396) in cerebral organoids with the APOE3Ch variant, consistent with the strikingly reduced tau pathology found in the resistant case. We identified Cadherin and Wnt pathways as signaling mechanisms regulated by the APOE3Ch variant through single cell RNA sequencing in cerebral organoids. We also identified elevated {beta}-catenin protein, a regulator of tau phosphorylation, as a candidate mediator of APOE3Ch resistance to tauopathy. Our findings show that APOE3Ch is necessary and sufficient to confer resistance to tauopathy in an experimental ex-vivo model establishing a foundation for the development of novel, protected case-inspired therapeutics for tauopathies, including Alzheimer's. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.12.523372v1?rss=1 Authors: Mendez-Vazquez, H., Roach, R. L., Nip, K., Sathler, M. F., Garver, T., Danzman, R. A., Moseley, M. C., Roberts, J. P., Koch, O. N., Steger, A. A., Lee, R., Arikkath, J., Kim, S. Abstract: {delta}-catenin is expressed in excitatory synapses and functions as an anchor for the glutamatergic AMPA receptor (AMPAR) GluA2 subunit in the postsynaptic density. The glycine 34 to serine (G34S) mutation in the{delta} -catenin gene is found in autism spectrum disorder (ASD) patients and induces loss of {delta}-catenin functions at excitatory synapses, which is presumed to underlie ASD pathogenesis in humans. However, how the G34S mutation causes loss of {delta}-catenin functions to induce ASD remains unclear. Here, using neuroblastoma cells, we discover that the G34S mutation generates an additional phosphorylation site for glycogen synthase kinase 3{beta} (GSK3{beta}). This promotes {delta}-catenin degradation and causes the reduction of {delta}-catenin levels, which likely contributes to the loss of {delta}-catenin functions. Synaptic {delta}-catenin and GluA2 levels in the cortex are significantly decreased in mice harboring the {delta}-catenin G34S mutation. The G34S mutation increases glutamatergic activity in cortical excitatory neurons while it is decreased in inhibitory interneurons, indicating changes in cellular excitation and inhibition. {delta}-catenin G34S mutant mice also exhibit social dysfunction, a common feature of ASD. Most importantly, inhibition of GSK3{beta} activity reverses the G34S-induced loss of {delta}-catenin function effects in cells and mice. Finally, using {delta}-catenin knockout mice, we confirm that {delta}-catenin is required for GSK3{beta} inhibition-induced restoration of normal social behaviors in {delta}-catenin G34S mutant animals. Taken together, we reveal that the loss of {delta}-catenin functions arising from the ASD-associated G34S mutation induces social dysfunction via alterations in glutamatergic activity and that GSK3{beta} inhibition can reverse {delta}-catenin G34S-induced synaptic and behavioral deficits. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.10.523392v1?rss=1 Authors: Pepe, A., Notario Manzano, R., Sartori-Rupp, A., Brou, C., Zurzolo, C. Abstract: Cell-to-cell communication it is a fundamental mechanism by which unicellular and multicellular organisms maintain relevant functions as development or homeostasis. Tunneling nanotubes (TNTs) are a type of contact-mediated cell-to-cell communication defined by being membranous structures based on actin that allow the exchange of different cellular material. TNTs have been shown to have unique structural features compared with other cellular protrusions and to contain the cell adhesion molecule N-Cadherin. Here, we investigated the possible role of N-Cadherin and of its primary linker to the actin cytoskeleton, alpha-Catenin in regulating the formation and transfer function of TNTs. Our data indicate that N-Cadherin through its downstream effector alpha-Catenin is a major regulator of TNT formation, ultrastructure, as well as of their ability to transfer material to other cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.13.520306v1?rss=1 Authors: Moparthi, L., Koch, S. Abstract: The Wnt/{beta}-catenin signaling pathway is a critical regulator of development and stem cell maintenance. Mounting evidence suggests that the context-specific outcome of Wnt signaling is determined by the collaborative action of multiple transcription factors, including members of the highly conserved forkhead box (FOX) protein family. However, the contribution of FOX transcription factors to Wnt signaling has not been investigated in a systematic manner. Here, we performed uniform gain-of-function screens of all 44 human FOX transcription factors to identify and classify new regulators of the Wnt/{beta}-catenin pathway. By combining {beta}-catenin reporter assays with Wnt pathway-focused qPCR arrays and proximity proteomics of selected FOX family members, we determine that most FOX proteins are involved in the regulation of Wnt pathway activity and the expression of Wnt ligands and target genes. Moreover, as a proof of principle we characterize class D and I FOX transcription factors as physiologically relevant positive and negative regulators of Wnt/{beta}-catenin signaling, respectively. We conclude that FOX proteins are common regulators of the Wnt/{beta}-catenin pathway that may control the outcome of Wnt signaling in a tissue-specific manner. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.05.519142v1?rss=1 Authors: Froehlich, J., Rose, K., Hecht, A. Abstract: Unrestrained transcriptional activity of {beta}-CATENIN and its binding partner TCF7L2 frequently underlies colorectal tumor initiation and is considered an obligatory oncogenic driver throughout intestinal carcinogenesis. Yet, the TCF7L2 gene carries inactivating mutations in about 10 % of colorectal tumors and is non-essential in colorectal cancer (CRC) cell lines. To determine whether CRC cells acquire TCF7L2-independence through cancer-specific compensation by other T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) family members, or rather lose addiction to beta-CATENIN/TCF7L2-driven gene expression altogether, we generated multiple CRC cell lines entirely negative for TCF/LEF or beta-CATENIN expression. Viability of these cells demonstrates complete beta-CATENIN- and TCF/LEF-independence, albeit one beta-CATENIN-deficient cell line eventually became senescent. Absence of TCF/LEF proteins and beta-CATENIN consistently impaired CRC cell proliferation, reminiscent of mitogenic effects of WNT/beta-CATENIN signaling in the healthy intestine. Despite this common phenotype, beta-CATENIN-deficient cells exhibited highly cell-line-specific gene expression changes with little overlap between beta-CATENIN- and TCF7L2-dependent transcriptomes. Apparently, beta CATENIN and TCF7L2 control sizeable fractions of their target genes independently from each other. The observed divergence of beta-CATENIN and TCF7L2 transcriptional programs, and the finding that neither beta-CATENIN nor TCF/LEF activity is strictly required for CRC cell survival has important implications when evaluating these factors as potential drug targets. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.514466v1?rss=1 Authors: Wang, Y., Mao, A., Liu, J., Li, P., Zheng, S., Tong, T., Li, Z., Zhang, H., Ma, L., Lin, J., Pang, Z., Han, Q., Li, F., Zhang, X., Chen, M., He, X., Zhang, X., Fei, T., Liu, B., Gao, D., Cao, L., Wang, Q., Li, Y., Sheng, R. Abstract: Wnt/{beta}-catenin signaling is a conserved pathway crucially governing development, tissue homeostasis and oncogenesis in metazoan. Through screening, we identified a deubiquitinase (DUB) USP10 as a novel modulator of Wnt/{beta}-catenin signaling. Mechanistically, USP10 binds to Axin1 via conserved motifs and stabilizes Axin1 through K48-linked deubiquitination. And in parallel, USP10 tethers Axin1 and {beta}-catenin physically, via stabilizing the phase separation of Axin1 through its intrinsically-disordered regions, which is regardless of its enzymatic activity. Functionally, we show USP10 prominently regulates zebrafish embryonic development and murine intestinal homeostasis by antagonizing Wnt/{beta}-catenin signaling. Additionally in human colorectal cancer, USP10 substantially represses cancer growth and correlates with Wnt/{beta}-catenin magnitude clinically. Collectively, we discovered USP10 functioning in multiple biological processes through repressing Wnt/{beta}-catenin signaling and unearthed a novel DUB-dependent and -independent dual-regulating mechanism by which USP10 utilizes in Wnt regulation context-dependently. Our study also suggested the potential of USP10 inhibitor in treating Wnt-related diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.23.513322v1?rss=1 Authors: Chen, X., Rong, K., Han, W., Pang, Y., Chai, G. Abstract: In previous research, miR-148a-3p deficiency was observed in bone malformation in hemifacial microsomia. Herein, in this article, we probed into the role of miR-148a-3p in bone physiology by utilizing miR-148a knock-out (KO) mice. Compared with wild-type (WT) or heterozygotic (HE) littermates, miR-148a knock-out mice manifested lower body weight, bone dysplasia with increased bone mass. Through in-vitro experiments, in terms of miR-148a-3p overexpression (miRNA mimic transfection) and knockout (primary cells from WT and KO littermates), we found that miR-148a-3p can suppress osteogenesis, either in the ALP activity or bone nodules formation. Afterward, by means of proteomics, combined with RNA-sequencing and prediction databases of microRNA targets (miRDB and TargetScan), nine candidate genes targeted by miR-148a-3p were identified. Among them, only Itga11 was regulated by mRNA degradation, while the others were modulated via post-transcriptional inhibition. Based on several online databases (GenePaint, BioGPS, STRING), Integrin Subunit Alpha 11 (Itga11) was suggested to play an essential role in osteogenesis and it was confirmed as one direct target of miR-148a-3p by dual-luciferase reporter assay. Meanwhile, gene set enrichment analysis (GSEA) indicated activation of PI3K-Akt signaling pathway and WNT signaling pathway in miR-148a KO mice. The thereafter western blot confirmed that PI3K/Akt/GSK3/{beta}-catenin signaling pathway was involved. Taken together, we demonstrated that miR-148a-3p can inhibit osteogenesis by targeting Itga11 via PI3K/Akt/GSK3/{beta}-catenin pathway. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.28.509312v1?rss=1 Authors: Patil, S., Chalkiadaki, K., Mergiya, T.-F., Simbriger, K., Amorim, I. S., Akerkar, S., Gkogkas, C. G., Bramham, C. R. Abstract: The mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is crucial for translation and regulated by Ser209 phosphorylation. However, the biochemical and physiological role of eIF4E phosphorylation in translational control of long-term synaptic plasticity is unknown. We demonstrate that phospho-ablated Eif4eS209A knockin mice are profoundly impaired in dentate gyrus LTP maintenance in vivo, while basal perforant path-evoked transmission and LTP induction are intact. mRNA cap-pulldown assays show that phosphorylation is required for synaptic activity-induced removal of translational repressors from eIF4E, allowing initiation complex formation. Using ribosome profiling, we identified selective, phospho-eIF4E-dependent translation of the Wnt signaling pathway in in vivo LTP. Surprisingly, the canonical Wnt effector, {beta}-catenin, was massively recruited to the eIF4E cap complex following LTP induction in wild-type, but not Eif4eS209A, mice. These results demonstrate a critical role for activity-evoked eIF4E phosphorylation in dentate gyrus LTP maintenance, bidirectional remodeling of the mRNA cap-binding complex, and mRNA-specific translational control linked to Wnt pathway. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.15.508086v1?rss=1 Authors: Parichha, A., Datta, D., Suresh, V., Chatterjee, M., Holtzman, M. J., Tole, S. Abstract: The dentate gyrus, a gateway for input to the hippocampal formation, arises from progenitors in the medial telencephalic neuroepithelium adjacent to the cortical hem. Dentate progenitors navigate a complex migratory path guided by two cell populations that arise from the hem, the fimbrial glia, and Cajal-Retzius (CR) cells. Since the hem expresses multiple Wnt genes, we examined whether {beta}-CATENIN, which mediates canonical Wnt signaling and also participates in cell adhesion, is necessary for the development of hem-derived lineages. We report that the fimbrial glial scaffold is disorganized and CR cells are mispositioned upon hem-specific disruption of {beta}-CATENIN. Consequently, the dentate migratory stream is severely affected, and the dentate gyrus fails to form. Using selective Cre drivers, we further determined that {beta}-CATENIN function is required in the fimbrial glial scaffold, but not in the CR cells, for guiding the dentate migration. Our findings highlight a primary requirement for {beta}-CATENIN for the organization of the fimbrial scaffold and a secondary role for this factor in dentate gyrus morphogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Study strengthens links between red meat and heart disease Queen Mary University (UK), 15 April 2021 An observational study in nearly 20,000 individuals has found that greater intake of red and processed meat is associated with worse heart function. The research is presented at ESC Preventive Cardiology 2021, an online scientific congress of the European Society of Cardiology (ESC).1 "Previous studies have shown links between greater red meat consumption and increased risk of heart attacks or dying from heart disease," said study author Dr. Zahra Raisi-Estabragh of Queen Mary University of London, UK.2,3 "For the first time, we examined the relationships between meat consumption and imaging measures of heart health. This may help us to understand the mechanisms underlying the previously observed connections with cardiovascular disease." The study included 19,408 participants of the UK Biobank.4 The researchers examined associations of self-reported intake of red and processed meat with heart anatomy and function. Three types of heart measures were analysed. First, cardiovascular magnetic resonance (CMR) assessments of heart function used in clinical practice such as volume of the ventricles and measures of the pumping function of the ventricles. Second, novel CMR radiomics used in research to extract detailed information from heart images such as shape and texture (which indicates health of the heart muscle). Third, elasticity of the blood vessels (stretchy arteries are healthier). The analysis was adjusted for other factors that might influence the relationship including age, sex, deprivation, education, smoking, alcohol, exercise, high blood pressure, high cholesterol, diabetes, and body mass index (BMI) as a measure of obesity. The researchers found that greater intake of red and processed meat was associated with worse imaging measures of heart health, across all measures studied. Specifically, individuals with higher meat intake had smaller ventricles, poorer heart function, and stiffer arteries - all markers of worse cardiovascular health. As a comparison, the researchers also tested the relationships between heart imaging measures and intake of oily fish, which has previously been linked with better heart health. They found that as the amount of oily fish consumption rose, heart function improved, and arteries were stretchier. Dr. Raisi-Estabragh said: "The findings support prior observations linking red and processed meat consumption with heart disease and provide unique insights into links with heart and vascular structure and function." The associations between imaging measures of heart health and meat intake were only partially explained by high blood pressure, high cholesterol, diabetes, and obesity. "It has been suggested that these factors could be the reason for the observed relationship between meat and heart disease," said Dr. Raisi-Estabragh. "For example, it is possible that greater red meat intake leads to raised blood cholesterol and this in turn causes heart disease. Our study suggests that these four factors do play a role in the links between meat intake and heart health, but they are not the full story." She noted that the study did not look into alternative mechanisms. But she said: "There is some evidence that red meat alters the gut microbiome, leading to higher levels of certain metabolites in the blood, which have in turn been linked to greater risk of heart disease." Dr. Raisi-Estabragh said: "This was an observational study and causation cannot be assumed. But in general, it seems sensible to limit intake of red and processed meat for heart health reasons."     More Fruits and Veggies Improves Sleep for Young Adults University of Michigan, April 15, 2021 Eating more fruits and vegetables can help young adults, especially young women, sleep better, a new study shows Young adults who reported eating less than five servings of fruits and vegetables per day reported a high prevalence of chronic insomnia symptoms, with over one-third reporting difficulties with falling asleep or maintaining sleep at least three times per week for three months or longer. Women who increased their fruit and vegetable intake by three or more servings over a three-month period were more than twice as likely to experience an improvement in these insomnia symptoms, according to the study in the Sleep Health Journal. “We were very excited to see that a fairly simple dietary intervention, such as encouraging an increase in fruit and vegetable consumption, could make such an impact on sleep,” says lead author Erica Jansen, research assistant professor of nutritional sciences at the University of Michigan’s School of Public Health. “We know from other literature that improving sleep improves overall quality of life and many other health outcomes, so the benefits likely extend beyond the sleep changes.” Jansen and senior author Gwen Alexander, a researcher in the public health sciences department at Henry Ford Health System, and colleagues analyzed data of more than 1,400 participants compiled by Detroit-based Henry Ford and the more rural Geisinger Health System headquartered in Danville, Pennsylvania. “From my health educator perspective, our study shows a link between dietary choices and improved sleep for young people who wish to improve their overall health and well-being,” Alexander says. “Our study was unique in that it investigated an understudied population of generally healthy young adults. Future research designed for this population has great potential to lead to better health habits.” Eligible young adults included those ages 21-30, who received any medical care at the centers and who reported eating less than five servings of fruits and vegetables per day. Researchers randomized the participants into one of three groups: one had an untailored web-based program to encourage higher fruits and vegetables consumption; the second had an age-targeted tailored web-based program; and the third group also included personalized e-coaching support. Young adults who increased their fruit and vegetable consumption by at least three servings experienced modest improvements in sleep latency (time to fall asleep) and insomnia over a three-month period, compared to participants with no change or smaller increases in fruits and vegetable intake, although there were no differences in sleep duration. Women who increased their fruit and vegetable intake by three or more servings reported a four-minute shorter time, on average, to fall asleep at follow-up, and twofold higher odds of improvement in insomnia symptoms. “What is unique about our study is that we were able to see that as fruit and vegetable intake changed, insomnia-related sleep characteristics also changed,” Jansen says. “We still cannot rule out that sleep characteristics changed first, which in turn caused a change in fruit and vegetable intake, but since the participants were part of a trial to increase fruit and vegetable intake, it is more likely the other way around. The participants were not told to change anything about their sleep habits.” The researchers hope the findings will be incorporated into other sleep hygiene principles, which include things like maintaining a consistent bedtime and rise time, eliminating screens prior to going to bed, sleeping in a dark, cool environment, and not drinking caffeine or alcohol before bed. Additional coauthors are from the University of South Carolina School of Medicine and the Henry Ford Health System.     Multivits, omega-3, probiotics, vitamin D may lessen risk of positive COVID-19 test British Medical Journal, April 20, 2021 Taking multivitamins, omega-3, probiotics or vitamin D supplements may lessen the risk of testing positive for SARS-CoV-2, the virus responsible for COVID-19 infection—at least among women—indicates a large population study, published online in the journal BMJ Nutrition Prevention & Health. But taking any of vitamin C, zinc, or garlic supplements wasn't associated with a lower risk of testing positive for the virus, the findings show. There has been plenty of celebrity endorsement of the use of dietary supplements to both ward off and treat COVID-19 infection since the start of the pandemic, note the researchers. In the UK alone, market share rose by 19.5% in the period leading up to the first national 'lockdown' on March 23 last year, with sales of vitamin C rising by 110% and those of multivits by 93%. Similarly, zinc supplement sales rose by 415% in the first week of March, at the height of COVID-19 fears in the U.S.. Dietary supplements can help to support a healthy immune system, but whether specific supplements might be associated with a lower risk of catching SARS-CoV-2 isn't known. In a bid to plug this knowledge gap, the researchers drew on adult users of the COVID-19 Symptom Study app to see if regular supplement users were less likely to test positive for SARS-CoV-2. The app was launched in the UK, the US, and Sweden in March 2020 to capture self-reported information on the evolution of the pandemic. Initially, it recorded the location, age and core health risk factors of its users. But as time went on, subscribers were asked to provide daily updates on a range of issues, including symptoms, coronavirus test results, and healthcare. People without obvious symptoms were also encouraged to use it. For the purposes of this study, the researchers analysed information supplied by 372,720 UK subscribers to the app about their regular use of dietary supplements throughout May, June, and July 2020 during the first wave of the pandemic as well as any coronavirus swab test results. Between May and July,175,652 UK subscribers regularly took dietary supplements;197,068 didn't. Around two thirds (67%) were women and over half were overweight (BMI of 27). In all, 23,521 people tested positive for SARS-CoV-2 and 349,199 tested negative between May and July. Taking probiotics, omega-3 fatty acids, multivits or vitamin D was associated with a lower risk of SARS-CoV-2 infection: by 14%, 12%, 13% and 9%, respectively, after accounting for potentially influential factors, including underlying conditions and usual diet. No such effects were observed among those taking vitamin C, zinc, or garlic supplements. And when the researchers looked specifically at sex, age and weight (BMI), the protective associations for probiotics, omega-3 fatty acids, multivits and vitamin D were observed only in women of all ages and weights. No such clear associations were seen in men. Despite some differences, the same overall patterns were mirrored in both the US (45,757) and Swedish (27,373) subscribers. The equivalent figures for the US and Sweden were a reduced risk of:18% and 37%, respectively for probiotics; 21% and 16%, respectively, for omega-3 fatty acids; 12% and 22%, respectively for multivits; and 24% and 19%, respectively, for vitamin D supplements. This is an observational study, and as such, can't establish cause. The researchers also acknowledge several limitations, including that the study relied on self reported data and a self selected group. No information was collected on supplement doses or ingredients either. But although the observed effects were modest, they were significant, note the researchers, who call for large clinical trials to inform evidence-based therapeutic recommendations. "We know that a range of micronutrients, including vitamin D, are essential for a healthy functioning immune system. This, in turn, is key to prevention of, and recovery from, infections. "But to date, there is little convincing evidence that taking nutritional supplements has any therapeutic value beyond maintaining the body's normal immune response," comments Professor Sumantra Ray, Executive Director, NNEdPro Global Centre for Nutrition and Health, which co-owns the journal. "What's more, this study wasn't primarily designed to answer questions about the role of nutritional supplements in COVID-19. This is still an emerging area of research that warrants further rigorous study before firm conclusions can be drawn about whether specific nutritional supplements might lessen the risk of COVID-19 infection," he cautions.   Vitamin D deficiency may impair muscle function Garvan Institute of Medical Research (Australia), April 16, 2021 Vitamin D deficiency may impair muscle function due to a reduction in energy production in the muscles, according to a mouse study published in the Journal of Endocrinology. Vitamin D deficient mice were found to have impaired muscle mitochondrial function, which may have implications for muscle function, performance and recovery. This may suggest that preventing vitamin D deficiency in older adults could help maintain better muscle strength and function and reduce age related muscle deterioration, but further studies are needed to confirm this.  Vitamin D is a hormone well known to be important for maintaining bone health and preventing rickets and osteoporosis. In recent years, vitamin D deficiency has been reported to be as prevalent as 40% in European populations and linked to increased risk for several conditions, including COVID-19, cancer and diabetes. Although these studies report association rather than causation, the benefits of vitamin D supplementation are now a major subject of health debate. Multiple studies have also linked low vitamin D levels to poor muscle strength, particularly in older people. Skeletal muscle enables us to move voluntarily and perform everyday activities. It is essential that they have enough energy to power these movements. Specialised organs in cells, called mitochondria, convert nutrients in to energy to meet this demand. Previous studies indicate that impaired muscle strength in people with vitamin D deficiency may be linked to impaired muscle mitochondrial function. Determining the role of vitamin D in muscle performance of older people is also difficult, as they may suffer from a number of pre-existing health conditions that can also affect their vitamin D status. Therefore, previous studies have been unable to determine how vitamin D may directly affect muscle performance. Dr Andrew Philp and his team at the Garvan Institute of Medical Research in Australia, and collaborating universities, used a mouse model to determine the effects of diet-induced vitamin D deficiency on skeletal muscle mitochondrial function in young, male mice. Mice were either fed a diet with normal quantities of vitamin D, or with no vitamin D to induce deficiency, for a period of 3 months. A typical vitamin D level for humans is 40-50 nmol.L-1, and acute vitamin D deficiency is diagnosed when levels drop below 12 nmol.L-1. On average, the mice in this study had vitamin D levels of 30 nmol.L1, with diet-induced vitamin D deficiency leading to levels of just 3 nmol.L-1. Although this level was more extreme than typically observed in people, it is still within the clinically-recognised range. Tissue and blood samples were collected monthly to quantify vitamin D and calcium concentrations and to assess markers of muscle mitochondrial function and number. After 3 months of diet-induced vitamin D deficiency skeletal muscle mitochondrial function was found to be impaired by up to 37%. This was not due to a reduced number of mitochondria or a reduction in muscle mass. "Our results show there is a clear link between vitamin D deficiency and oxidative capacity in skeletal muscle. They suggest that vitamin D deficiency decreases mitochondrial function, as opposed to reducing the number of mitochondria in skeletal muscle." Dr Philp comments. "We are particularly interested to examine whether this reduction in mitochondrial function may be a cause of age related loss in skeletal muscle mass and function." These findings suggest that vitamin D deficiency may impair mitochondrial function and reduce the amount of energy produced in the muscles, which may lead to poor muscle function. Therefore, preventing vitamin D deficiency in older people may help maintain muscle performance and reduce the risk of muscle related diseases, such as sarcopenia. However, further studies that investigate the direct effect of vitamin D deficiency on muscle function and strength are necessary to confirm this. Whilst this study indicates that vitamin D deficiency can alter mitochondrial function in skeletal muscle, Dr Philp and his team were unable to determine precisely how this process occurred. Therefore, their future work aims to establish how vitamin D deficiency alters mitochondrial control and function in skeletal muscle.     Psychedelic experience may not be required for psilocybin's antidepressant-like benefits So-called 'magic mushroom' drug seems to work through multiple brain mechanisms for its different effects University of Maryland School of Medicine, April 16, 2021 University of Maryland School of Medicine (UMSOM) researchers have shown that psilocybin--the active chemical in "magic mushrooms"-- still works its antidepressant-like actions, at least in mice, even when the psychedelic experience is blocked. The new findings suggest that psychedelic drugs work in multiple ways in the brain and it may be possible to deliver the fast-acting antidepressant therapeutic benefit without requiring daylong guided therapy sessions. A version of the drug without, or with less of, the psychedelic effects could loosen restrictions on who could receive the therapy, and lower costs, making the benefits of psilocybin more available to more people in need. In all clinical trials performed to date, the person treated with psilocybin remains under the care of a guide, who keeps the person calm and reassures them during their daylong experience. This can include hallucinations, altered perception of time and space, and intense emotional and spiritual encounters.  Researchers in the field have long attributed psilocybin's effectiveness to the intense psychedelic experience.  "We do not understand the mechanisms that underlie the antidepressant actions of psilocybin and the role that the profound psychedelic experience during these sessions plays in the therapeutic benefits," says Scott Thompson, Ph.D., Professor and Chair, Department of Physiology at UMSOM and senior author of the study. "The psychedelic experience is incredibly powerful and can be life-changing, but that could be too much for some people or not appropriate."  Several barriers prevent the wide-spread use of psychedelic compounds. For example, there is fear that the psychedelic experience may promote psychosis in people who are predisposed to severe mental disorders, like bipolar disorder and schizophrenia, so the clinical therapy sessions performed to-date have been limited to a highly selected screened group without a family history of these disorders.  Dr. Thompson adds that there may also be an equity issue because not everyone can take several days off work to prepare and engage in the experience. The costs of staffing a facility with at least one trained guide per treated person per day and a private space may also be prohibitive to all but a few. He says it is conceivable that a depression treatment derived from psilocybin could be developed without the psychedelic effects so people can take it safely at home without requiring a full day in a care facility. For their study, led by UMSOM MD/PhD student Natalie Hesselgrave, the team used a mouse model of depression in which mice were stressed for several hours a day over 2-3 weeks. Because researchers cannot measure mouse moods, they measure their ability to work for rewards, such as choosing to drink sugar water over plain water. People suffering from depression lose the feeling of pleasure for rewarding events. Similarly, stressed mice no longer preferred sugar water over plain water. However, 24 hours after a dose of psilocybin, the stressed mice regained their preference for the sugar water, demonstrating that the drug restored the mice's pleasure response.  Psilocybin exerts its effects in people by binding to and turning on receptors for the chemical messenger serotonin. One of these receptors, the serotonin 2A receptor, is known to be responsible for the psychedelic response. To see if the psychedelic effects of psilocybin were needed for the anti-depressive benefits, the researchers treated the stressed mice with psilocybin together with a drug, ketanserin, which binds to the serotonin 2A receptor and keeps it from being turned on. The researchers found that the stressed mice regained their preference for the sugar water in response to psilocybin, even without the activation of the psychedelic receptor. "These findings show that activation of the receptor causing the psychedelic effect isn't absolutely required for the antidepressant benefits, at least in mice," says Dr. Thompson, "but the same experiment needs to be performed in depressed human subjects." He says his team plans to investigate which of the 13 other serotonin receptors are the ones responsible for the antidepressant actions. "This new study has interesting implications, and shows that more basic research is needed in animals to reveal the mechanisms for how these drugs work, so that treatments for these devastating disorders can be developed" says Albert E. Reece, MD, PhD, MBA, Executive Vice President for Medical Affairs, University of Maryland Baltimore, and the John Z. and Akiko K. Bowers Distinguished Professor and Dean, University of Maryland School of Medicine.   Tea compound promotes formation of osteoblasts under inflammatory environment and increases bone mass First Affiliated Hospital of Soochow University (China), April 7, 2021   According to news originating from Suzhou, People’s Republic of China, the research stated, “Postmenopausal osteoporosis is a disease of bone mass reduction and structural changes due to estrogen deficiency, which can eventually lead to increased pain and fracture risk.” Our news correspondents obtained a quote from the research from First Affiliated Hospital of Soochow University: “Chronic inflammatory microenvironment leading to the decreased activation of osteoblasts and inhibition of bone formation is an important pathological factor that leads to osteoporosis. Theaflavin-3,3’-digallate (TFDG) is an extract of black tea, which has potential anti-inflammatory and antiviral effects. In our study, we found that TFDG significantly increased the bone mass of ovariectomized (OVX) mice by micro-CT analysis. Compared with OVX mice, TFDG reduced the release of proinflammatory cytokines and increased the expression of osteogenic markers in vivo. In vitro experiments demonstrated that TFDG could promote the formation of osteoblasts in inflammatory environment and enhance their mineralization ability. In this process, TFDG activated MAPK, Wnt/b-Catenin and BMP/Smad signaling pathways inhibited by TNF-a, and then promoted the transcription of osteogenic related factors including Runx2 and Osterix, promoting the differentiation and maturation of osteoblasts eventually.” According to the news reporters, the research concluded: “In general, our study confirmed that TFDG was able to promote osteoblast differentiation under inflammatory environment, enhance its mineralization ability, and ultimately increase bone mass in ovariectomized mice. These results suggested that TFDG might have the potential to be a more effective treatment of postmenopausal osteoporosis.”     Patients who are overweight or obese at risk of more severe COVID-19   Murdoch Children's Research Institute and University of Queensland, April 16, 2021   Patients who are overweight or obese have more severe COVID-19 and are highly likely to require invasive respiratory support, according to a new international study.  The research, led by the Murdoch Children's Research Institute (MCRI) and The University of Queensland and published in Diabetes Care, found obese or overweight patients are at high risk for having worse COVID-19 outcomes. They are also more likely to require oxygen and invasive mechanical ventilation compared to those with a healthy weight.  MCRI researcher Dr Danielle Longmore said the findings, which highlighted the relationship between obesity and increased COVID-19 disease burden, showed the need to urgently introduce strategies to address the complex socio-economic drivers of obesity, and public policy measures such as restrictions on junk food advertising.  "Although taking steps to address obesity in the short-term is unlikely to have an immediate impact in the COVID-19 pandemic, it will likely reduce the disease burden in future viral pandemics and reduce risks of complications like heart disease and stroke," she said. The study looked at hospitalised SARS-CoV-2 patients from 18 hospitals in 11 countries including China, America, Italy, South Africa and The Netherlands.  Among the 7244 patients aged 18 years and over, 34.8 per cent were overweight and 30.8 per cent were obese.  COVID-19 patients with obesity were more likely to require oxygen and had a 73 per cent greater chance of needing invasive mechanical ventilation. Similar but more modest results were seen in overweight patients. No link was found between being overweight or obese and dying in hospital from COVID-19.  Cardiovascular and pre-existing respiratory diseases were associated with increased odds of in-hospital deaths but not a greater risk for needing oxygen and mechanical ventilation. For patients with pre-existing diabetes, there was increased odds of needing invasive respiratory support, but no additionally increase in risk in those with obesity and diabetes.  Men were at an increased risk of severe COVID-19 outcomes and needing invasive mechanical ventilation. In those aged over 65 years, there was an increased chance of requiring oxygen and higher rates of in-hospital deaths. The University of Queensland's Dr Kirsty Short, who co-led the research, said almost 40 per cent of the global population was overweight or obese.  "Obesity is associated with numerous poor health outcomes, including increased risk of cardiometabolic and respiratory disease and more severe viral disease including influenza, dengue and SARS-CoV-1," she said.  Dr Short said while previous reports indicated that obesity was an important risk factor in the severity of COVID-19, almost all this data had been collected from single sites and many regions were not represented. Moreover, there was a limited amount of evidence available about the effects of being overweight or obese on COVID-19 severity.  "Given the large scale of this study we have conclusively shown that being overweight or obese are independent risk factors for worse outcomes in adults hospitalised with COVID-19," she said. MCRI Professor David Burgner, who co-led the research, said the data would help inform immunisation prioritisation for higher-risk groups. "At the moment, the World Health Organization has not had enough high-quality data to include being overweight or obese as a risk factor for severe COVID-19 disease. Our study should help inform decisions about which higher-risk groups should be vaccinated as a priority," he said.   Neuroprotective Herbs for the Management of Alzheimer’s Disease University of Central Florida and University of California, Los Angeles   Background—Alzheimer’s disease (AD) is a multifactorial, progressive, neurodegenerative disease that is characterized by memory loss, personality changes, and a decline in cognitive function. While the exact cause of AD is still unclear, recent studies point to lifestyle, diet, environmental, and genetic factors as contributors to disease progression. The pharmaceutical approaches developed to date do not alter disease progression. More than two hundred promising drug candidates have failed clinical trials in the past decade, suggesting that the disease and its causes may be highly complex. Medicinal plants and herbal remedies are now gaining more interest as complementary and alternative interventions and are a valuable source for developing drug candidates for AD. Indeed, several scientific studies have described the use of various medicinal plants and their principal phytochemicals for the treatment of AD. This article reviews a subset of herbs for their anti-inflammatory, antioxidant, and cognitive-enhancing effects. Methods—This article systematically reviews recent studies that have investigated the role of neuroprotective herbs and their bioactive compounds for dementia associated with Alzheimer’s disease and pre-Alzheimer’s disease. PubMed Central, Scopus, and Google Scholar databases of articles were collected, and abstracts were reviewed for relevance to the subject matter. Conclusions—Medicinal plants have great potential as part of an overall program in the prevention and treatment of cognitive decline associated with AD. It is hoped that these medicinal plants can be used in drug discovery programs for identifying safe and efficacious small molecules for AD.   1.1. Ashwagandha (Withania somnifera) Ashwagandha, commonly called Indian ginseng or winter cherry, is one of the most prominent herbs prescribed as a brain rejuvenator for AD. It is prescribed to increase energy, improve overall health and longevity, and as a nerve tonic [86]. Ashwagandha has been shown to possess antioxidant activity, free radical scavenging activity, as well as an ability to support a healthy immune system [87]. Ashwagandha contains several bioactive compounds of great interest, such as ergostane-type steroidal lactones, including withanolides A-Y, dehydrowithanolide-R, withasomniferin-A, withasomidienone, withasomniferols A-C, withaferin A, withanone, and others. Other constituents include the phytosterols sitoindosides VII-X and beta-sitosterol and alkaloids [86,88]. A subset of these components has been shown to scavenge free radicals generated during the initiation and progression of AD. Molecular modeling studies showed that withanamides A and C uniquely bind to the active motif of Aβ25-35 and prevent fibril formation. Furthermore, these compounds protected PC-12 cells and rat neuronal cells from β-amyloid-induced cell death [89,90,91]. Treatment with the methanol extract of ashwagandha triggered neurite outgrowth in a dose- and time-dependent manner in human neuroblastoma cells [29], and, in another study involving cultured rat cortical neurons, treatment with Aβ peptide induced axonal and dendritic atrophy and loss of pre-and postsynaptic stimuli [92]. Subsequent treatment with withanolide A induced significant regeneration of both axons and dendrites and restored the pre- and post-synapses in the cultured cortical neurons. In vivo, withanolide A inhibited Aβ(25–35)-induced degeneration of axons, dendrites, and synapses in the cerebral cortex and hippocampus and also restored Aβ-peptide-induced memory deficits in mice [93]. The in vivo ameliorative effects were maintained even after the discontinuation of the drug administration. Aqueous extracts of ashwagandha increased acetylcholine (ACh) content and choline acetyl transferase activity in rats, which might partly explain the cognition-enhancing and memory-improving effects [29,94,95]. Treatment with the root extract caused the upregulation of the low-density lipoprotein receptor-related protein, which enhanced the Aβ clearance and reversed the AD pathology in middle-aged and old APP/PS1 mice [96]. Oral administration of a semi-purified extract of ashwagandha reversed behavioral deficits and blocked the accumulation of Aβ peptides in an APP/PS1 mouse model of AD. This therapeutic effect of ashwagandha was mediated by the liver low-density lipoprotein receptor-related protein [96]. Using an AD model of Drosophila melanogaster, researchers noted that treatment with ashwagandha mitigated Aβ toxicity and also promoted longevity [97]. Despite the extensive literature on the therapeutic effects of ashwagandha, there are limited data on its clinical use for cognitive impairment [98]. In a prospective, randomized, double-blind, placebo-controlled pilot study involving 50 subjects with mild cognitive impairment, subjects were treated with either ashwagandha root extract (300 mg twice daily) or placebo for eight weeks. After eight weeks of study, the ashwagandha treatment group demonstrated significant improvements in both immediate and general memory tests compared to the placebo group. Furthermore, the treatment group showed significant improvement in executive function, sustained attention, and information-processing speed [99]. These studies lend credence to ashwagandha’s role in enhancing memory and improving executive function in people with SCI or MCI. 1.2. Brahmi (Bacopa monnieri) Brahmi, or Bacopa monnieri (Bm), is a perennial creeper medicinal plant found in the damp and marshy wetlands of Southern and Eastern India, Australia, Europe, Africa, Asia, and North and South America. In the Ayurvedic system of medicine, Bm is recommended for mental stress, memory loss, epilepsy, insomnia, and asthma [34,36]. The bioactive phytochemicals present in this plant include saponins, bacopasides III, IV, V, bacosides A and B, bacosaponins A, B, C, D, E, and F, alkaloids, sterols, betulic acid, polyphenols, and sulfhydryl compounds, which may be responsible for the neuroprotective roles of the plant. Both in vitro and in vivo studies show that these phytochemicals have an antioxidant and free radical scavenging action by blocking lipid peroxidation in several areas of the brain [36,100,101,102]. Bm acts by reducing divalent metals, scavenging reactive oxygen species, decreasing the formation of lipid peroxides, and inhibiting lipoxygenase activity [103]. Numerous studies have also shown Bm’s role in memory and intellect [33,56,100,104,105,106]. To determine the neuroprotective effect of Bm in a rat model of AD, researchers tested an alcoholic extract of Bm at doses of 20, 40, and 80 mg/kg for a period of 2 weeks before and 1 week after the intracerebroventricular (icv) administration of ethylcholine aziridinium ion (AF64A). Spatial memory was tested using the Morris water maze (MWM), and the cholinergic neuron density was determined using histological techniques. The researchers showed that Bm extract improved the escape latency time in the MWM test and blocked the reduction of cholinergic neuron densities [35]. Another group reported the reversal of colchicine-induced cognitive deficits by a standardized extract of Bm. In addition to reversing colchicine-triggered cognitive impairment, the Bm extract also attenuated colchicine-induced oxidative damage by decreasing the protein carbonyl levels and restoring the activities of the antioxidant enzymes [107]. Most of the studies exploring the cognitive-enhancing effects of Bm in humans focused on normal, aged individuals. In a double-blind, randomized, placebo-controlled trial on 35 individuals aged above 55 years, subjects received either 125 mg of Bm extract or a placebo twice a day for a period of 12 weeks, followed by a placebo period of another four weeks. Subjects underwent a battery of memory tests, including general information, orientation, mental control, logical memory, digit forward, digit backward, visual reproduction, and paired association learning. Subjects were scored on each sub-test, and total memory score was calculated by adding the score of all subtests. A significant improvement was observed in mental control, logical memory, and paired association learning in Bm-treated patients compared to the placebo group at 8 and 12 weeks after initiation of the trial [37]. The results suggested the use of Bm in the treatment of age-associated memory impairment. Ten subjects were given 500 mg of Sideritis extract, 320 mg Bm extract, or a combination using a crossover design. Sideritis extract is rich in a variety of flavonoids and has been shown to improve cognition in animal models of AD [108]. The Attention d2 Test is a neuropsychological measure of selective and sustained attention and visual scanning speed. Assessment tests revealed that Sideritis extract combined with a low-dose Bm extract resulted in improvement in the d2 concentration test score [109]. A similar effect of Bm alone was observed only after repetitive dosing, suggesting that the long-term memory effects seen with repetitive dosing of Bm may be a promising therapeutic option for subjects suffering from MCI [109]. In another prospective, non-comparative, multicenter trial involving 104 subjects who suffered from MCI, Bm extract in combination with astaxanthin, phosphatidylserine, and vitamin E was given for 60 days. The tested combination formula was well tolerated. Cognitive and mnemonic performance was assessed with validated instruments including Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-cog) and Clock-Drawing Test (CDT) that can assess the risk of MCI progression to AD. Researchers noted significant improvements in ADAS-cog and CDT scores [110]. The observed sixty-day improvements in ADAS-cog and CDT were statistically significant as compared with baseline values. Memory is affected by several factors, including focus and attention, neurotransmitters, hormones, trophic factors, cyclic AMP, ion channels, protein transcription, synapse formation, and nutrients. Some of these processes can be modulated by Bm extract alone or in combination with other compounds. The abovementioned study design is similar to our therapeutic program for people with SCI and MCI, where Bm is administered in combination with other nutraceuticals and cogniceuticals [15,111]. 1.3. Cat’s Claw (Uncaria tomentosa) Cat’s claw (CC) is a tropical vine with hooked thorns that resemble the claws of a cat and is mainly recommended for its potential role in the treatment of AD and pre-AD. It is found mainly in the Amazon rainforest and other areas of South and Central America. This medicinal plant contains oxindole alkaloids, polyphenols (flavonoids, proanthocyanidins, and tannins), glycosides, pentacyclic alkaloids, and sterols [38,39]. CC is known for its immune-modulating and anti-inflammatory effects and for its role as a free radical scavenger. Based on in vitro studies, the anti-inflammatory effect of CC is attributed to its ability to inhibit iNOS gene expression, nitrate formation, cell death, PGE2 production, and the activation of NF-κB and TNF-α [45]. Using a transgenic mouse model of Alzheimer’s disease, a significant reduction in the Aβ load (by 59%) and plaque number (by 78%) in the hippocampus and cortex was observed after treating 8-month-old mice with the CC extract for 14 days [44]. CC extract also caused a significant reduction in astrocytosis and microgliosis, and it improved hippocampus-dependent memory. Some of the components in the CC extract crossed the blood–brain barrier (BBB) and entered the brain parenchyma following intravenous injection [44]. Pre-clinical studies suggest that CC extract inhibits the formation of plaques and tangles, reduces astrocytosis and microgliosis and improves memory in mouse models of AD [43,44]. CC extract not only prevented the formation and aggregation of Aβ fibrils and tau protein paired helical filaments, but it also facilitated the disaggregation of preformed fibrils and tau protein tangles [43,44]. While proanthocyanidin B2 was identified as the primary phytochemical with plaque-and tangle-dissolving activity, other polyphenols present in the CC extract also possess plaque-reducing activity [44]. Based on pre-clinical studies, Cat’s claw may be effective for memory loss and cognitive decline associated with AD, although no studies have been carried out in humans. 1.4. Ginkgo Biloba Ginkgo biloba (Gb) has been in the spotlight primarily for its potential role in treating AD. Gb also appears promising as a therapeutic agent for several other chronic and acute forms of diseases. The main pharmacologically active groups of compounds are flavonoids and terpenoids. Almost all clinical studies use Gb extract that contains a combination of flavonoid glycosides, terpene lactones, and ginkgolic acids [50]. Gb extract has shown beneficial effects in treating Alzheimer’s, cardiovascular diseases, cancer, tinnitus, and other age-associated conditions [49,50]. The suggested mechanisms of the Gb extract are its antioxidant effect, anti-platelet activating factor activity for vascular diseases, inhibition of β-amyloid peptide aggregation in AD, and decreased expression of peripheral benzodiazepine receptor for stress alleviation [48,49,50]. Gb is popular as a treatment for early-stage AD and vascular dementia. Gb extract reverses β-amyloid and NO-induced toxicity in vitro and reduces apoptosis both in vitro and in vivo [112,113,114]. Treatment with Gb extract enhanced memory retention in young and old rats and improved short-term memory in mice [49,115]. Several studies indicate that ginkgo delays the progression of AD and is as effective as the cholinesterase inhibitors for treating AD. A modest improvement in cognitive function was observed in AD subjects in various randomized, double-blind, placebo-controlled trials [116,117,118]. Gb extract also improves ADLs among AD individuals and is preferred over other AD medications because of its negligible adverse effects [119,120]. 1.5. Gotu Kola (Centella asiatica) Considered both a nutraceutical and cogniceutical, Gotu kola (Gk) is a staple in Chinese, Indonesian, and Ayurvedic medicine [57]. This medicinal plant is used to strengthen the brain, heal skin issues, and promote liver and kidney health. Gk is considered a rejuvenating herb for nerve and brain cells as it is believed to promote intelligence and improve memory [54,55,56,57]. In vitro studies using various Gk plant derivatives (asiaticosides, asiatic acid, madecassoside, and madasiatic acid) showed that these compounds were capable of blocking H2O

Rahul Aras, CEO, Iterion Therapeutics, describes their lead product Tegavivint, a potent selective inhibitor of nuclear beta-catenin, a high-value oncology target because of its role in cell proliferation, differentiation, immune evasion and other aspects of cancer development. An early clinical study in patients with the rare disease Desmoid tumors has showed that their tumor stabilized and reversed in size.  Progress is being driven by recent understanding of the role of TBL1 in driving oncogenic activity of beta-catenin. #IterionTherapeutics #Tegavivint #cancer #DesmoidTumors #nuclearbetacatenin #TBL1 Iteriontherapeutics.com Listen to the podcast here  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.25.354415v1?rss=1 Authors: Heier, J. A., Pokutta, S., Dale, I. W., Kim, S. K., Hinck, A. P., Weis, W. I., Kwiatkowski, A. V. Abstract: Alpha-catenin binds directly to beta-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates alpha-catenin conformation: actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. Here we describe the biochemical properties of alpha-T(testes)-catenin, an alpha-catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that alpha-T-catenin binds the beta-catenin/N-cadherin complex with a similar low nanomolar affinity to that of alpha-E-catenin. Limited proteolysis revealed that the alpha-T-catenin M-region adopts a more open conformation than alpha-E-catenin. The alpha-T-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated alpha-T-catenin M-region is not autoinhibited and thereby distinct from alpha-E-catenin. However, the alpha-T-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates M-region binding to vinculin. In cells, alpha-T-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results indicate that the alpha-T-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of alpha-T-catenin allow it to function as a scaffold for building specific adhesion complexes. Copy rights belong to original authors. Visit the link for more info

Rahul Aras, CEO, Iterion Therapeutics, describes their lead product Tegavivint, a potent selective inhibitor of nuclear beta-catenin, a high-value oncology target because of its role in cell proliferation, differentiation, immune evasion and other aspects of cancer development. An early clinical study in patients with the rare disease Desmoid tumors has showed that their tumor stabilized and reversed in size.  Progress is being driven by recent understanding of the role of TBL1 in driving oncogenic activity of beta-catenin. #IterionTherapeutics #Tegavivint #cancer #DesmoidTumors #nuclearbetacatenin #TBL1 Iteriontherapeutics.com Download the transcript here  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.14.297283v1?rss=1 Authors: Biswas, S., Emond, M. R., Chenoweth, K. P., Jontes, J. Abstract: The proliferation of neural progenitor cells provides the cellular substrate from which the nervous system is sculpted during development. The {delta}-protocadherin family of homophilic cell adhesion molecules is essential for the normal development of the nervous system and has been linked to an array of neurodevelopmental disorders. However, the biological functions of {delta}-protocadherins are not well-defined. Here, we show that the {delta}-protocadherins regulate proliferation in neural progenitor cells, as lesions in each of six, individual {delta}-protocadherin genes increase cell division in the developing hindbrain. Moreover, Wnt/{beta}-catenin signaling is upregulated in {delta}-protocadherin mutants and inhibition of the canonical Wnt pathway occludes the observed proliferation increases. We show that the {delta}-protocadherins physically associate with the Wnt receptor Ryk, and that Ryk is required for the increased proliferation in protocadherin mutants. Thus, the {delta}-protocadherins act as novel regulators of Wnt/{beta}-catenin signaling during neural development and could provide lineage-restricted local regulation of canonical Wnt signaling and cell proliferation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.12.092148v1?rss=1 Authors: Wang, J., Jiang, J., Yang, X., Wang, L., Xiao, B. Abstract: The mechanically activated Piezo channel plays a versatile role in conferring mechanosensitivity to various cell types. However, how it incorporates its intrinsic mechanosensitivity and cellular components to effectively sense long-range mechanical perturbation across a cell remains elusive. Here we show that Piezo1 is biochemically and functionally tethered to the actin cytoskeleton via the E-cadherin-{beta}-catenin mechanotransduction complex, whose perturbation significantly impairs Piezo1-mediated responses. Mechanistically, the adhesive extracellular domain of E-cadherin interacts with the cap domain of Piezo1 that controls the transmembrane gate, while its cytosolic tail might interact with the cytosolic domains of Piezo1 that are in close proximity to its intracellular gates, allowing a direct focus of adhesion-cytoskeleton-transmitted force for gating. Specific disruption of the intermolecular interactions prevents cytoskeleton-dependent gating of Piezo1. Thus, we propose a force-from-filament model to complement the previously suggested force-from-lipids model for mechanogating of Piezo channels, enabling them to serve as versatile and tunable mechanotransducers. Copy rights belong to original authors. Visit the link for more info

ONCE UPON A GENE - EPISODE 021 Healthier Healthcare for All Christie Olson is interviewing me and we’re firing up a conversation about the healthcare challenges along my journey with Ford in hopes that it shines a light on some of these issues that parents face regularly.   EPISODE HIGHLIGHTS Effie, introduce the Once Upon a Gene podcast.  On October 31st, 2019 I launched my podcast, which I started in order to have conversations about raising children with disabilities and rare disease. I wanted to help create a community for parents with families like mine.  Let’s talk about how we know each other and how we met. At our mutual favorite place, Kindering,our local birth to three center, there’s a program for kids around 16 months old where you can bring them out of the home setting and bring them into a mini preschool setting where the parents come to the class as well. We met because your son, Peter was in the same class as Ford for a few months.  Introduce you son, Ford.  Ford is my favorite subject. He’s the brightest light, so hard-working, so smart and so funny. His laugh is constantly in my head- I hear it all the time. He has this spirit about him that brings you down to Earth, He loves the comfort of his home and his stuff and being with family. He loves weather, being outside and the wind on his face. He’s really easy going and he just never gives up. He’s the hardest working person I’ve ever met.  Talk about when Ford began receiving outpatient therapies from Kindering. Ford was admitted into Seattle Children’s at about three months old for failure to thrive. When we left after about a week, I think they set things in motion for us. I don’t recall making those calls, although maybe I did. I think Seattle Children’s played a huge part in connecting me to Kindering. They set up an evaluation for Ford and I brought him in and theses two therapists did a few different things with Ford. I was really emotional and not really understanding the scope of what was going on with Ford at the time and they were so calm with me and so patient. They confirmed that Ford definitely needed therapy services, that he qualified and that they would contact me. When they contacted me, they also said Ford qualified for home visits, which was the biggest relief. We were set up with speech therapy, physical therapy, occupational therapy and vision therapy. We then found our footing with which ones we needed to focus on more.  Can you tell me about what Ford’s diagnosis is? Ford was diagnosed with CTNNB1, which is a gene that mutated. It’s a random occurrence and not something that Casey or I passed down to Ford. That gene specifically is in charge of producing a protein called Catenin beta-1 and has lots of jobs that has to do with cell growth, reproduction, cell adhesion and it affects many parts of Ford’s body. There’s cognitive disabilities, motor disabilities and speech disabilities. Ford isn’t able to sit or walk yet and he’s non-verbal. It creates a lot of difficulty in getting around and doing daily tasks. Random things also affected include his vision and a sensitivity to the sun. Most of the children diagnosed are really young, so we don’t have a lot of knowledge about what can happen later. CTNNB1 was found in 2012 and there are fewer than 200 people with the diagnosis so there’s a ton we don’t know yet. We’re growing a lot with the access to genetic testing, so hopefully we can keep learning more and connect it to similar diagnoses. Are there adults with this mutation? There are about three people older than 18. Yes, there are probably hundreds throughout the world who have never been diagnosed or who have been mis-diagnosed with Cerebral Palsy. If someone were tested before 2012, they wouldn’t have been tested and wouldn’t have been diagnosed. That’s the case for a lot of rare diseases where the science, innovation and genetic testing is coming out so fast that anything prior to that, a person would never know unless they were re-tested. Were there any challenges or difficulties that you had accessing the genetic testing you needed? At the time, I didn’t know what genetic testing was or that we needed it. We got referred to a geneticist by our pediatrician. She thought she knew what Ford had, but she wanted him to get a full exome sequence because she was positive that something genetic was underlying for Ford. I’m thankful that she took the initiative to do that so early on because I see so many families who’s doctor has never even brought it up and they learn years later from other parents to ask for it.  Discuss what a typical day with Ford looks like. The first thing we do is give Ford his medications. He takes medicine for dystonia, which is the same medication that someone with Parkinson’s would take. After he has had his medicine, we hug, I open the window and we look outside. I get him dressed and get his food ready. Ford’s muscle tone also prevents him from swallowing well and he’s never been able to really get a meal down without gagging or vomiting. He’s g-tube fed, so I get his food ready, plug it in to his tummy and cross my fingers for about an hour and a half that it doesn’t come back up. Then I spend a few minutes getting his foot braces and shoes on, which are tough to get on.  Is there certain equipment or supplies you’ve had to make work for you?  One of the biggest challenges that I had in the beginning was finding a way for Ford to sit. There are zero things on the market for a child who has low muscle tone that are under $1000. I spent so much money trying to find a way for Ford to sit, to sit safely and in a position for his body to do its jobs- and nothing really worked. This amazing woman named Trish at Kindering made a chair at her house with wood and screws. She made Ford a chair just for his body and we used that for two years until he grew out of it. Have you found there’s any insurance reimbursement for equipment?  Medical insurance itself is a huge challenge. Eventually I was able to get a feeding activity chair for Ford. It took me about a year to get it and I still use it today. It takes a long time to get things like that approved, so you have to figure something else out in the meantime. You can wait a really long time and go through appeals and denials for a long time. And you have to actually have to know about it. My therapist didn’t recommend it or mention it. I actually saw other people using it before I even knew something like that existed.  What are the technicalities of the g-tube and feeding pump you guys use?  For the first few months, when Ford was very young, he used an ng-tube which went through his nose. His g-tube was placed, which is sewn into his belly and there’s a manually pump that goes with it. Some kids use a pump, some don’t and some the food is administered with a syringe. He receives 100% of his nutrition from the g-tube. It’s simple to use once you have done it a couple times. You measure the food, pour it in the bag, click it into his stomach with an extension that goes from the tube to the bag and you press play. Ford uses an infinity pump which seems like the easiest pump to use from what I’ve seen. It’s still a struggle for myself and Ford’s providers to determine what Ford will tolerate as far as rates and amounts. I think it has to do with Ford’s condition and we haven’t figured out the why or how to master it. He doesn’t gain weight much, he’s been the same weight for the last two years- maybe gained a pound. We’re constantly worried about his nutrition and whether or not he’s getting enough calories. What do family meals and holidays look like even though Ford’s not eating food at the table?   That’s not as hard as it used to be. We have a chair that Ford sits in at the grandparent’s homes. He sits in his chair and we plug him up to his g-tube at the same time and most times, if not always, Ford gets to have his ipad while we’re at the dinner table and he gets to watch videos. Sometimes he wants a taste of food, which is exciting. Sometimes he actually can participate, but he mainly just wants to be around the family. Everyone is good about making Ford feel like he’s at the table. He joins us, just differently than most kids.  Does Ford utilize an ipad for communication? We recently got an application called Proloquo2Go to go which we’re all still learning. Ford thinks it’s funny because he can push all the buttons and it speaks back to him. Ford’s means of communication for other people will probably largely be this app. It’s an amazing resource and Ford’s responding to it.  For people who work in healthcare, how can we come alongside you and provide you tangible help and validation? What would have been really helpful for me in the beginning is a person or packet with every resource I would need- a comprehensive take home packet to know next steps. Having this packet would have been helpful to know where I was supposed to go. In appointment settings in general, the empathy from providers matters. It matters that we feel listened to, that they know the diagnosis and have read the chart before coming into the room, that they think outside the box. Including us in the care plan as a teammate would be awesome, but I know that comes down to how long a doctor has to prepare for a visit. I want doctors to give me hope and encouragement, motivation and I want them to have a positive outlook on their future- just being on my team on a human level and not just my doctor. It would also be helpful if there were better collaboration between providers.  What are things that you appreciate most about the healthcare system? I’m appreciative for having healthcare. I’m grateful for the nurses involved in healthcare. They’re such a beautiful bridge for patients and families. They’re so helpful, empathetic, a lot of them go out of their way to make sure you know about something or to give you something that you can take home and find useful for your child. I’m grateful for doctors who take the initiative and want to seek out a diagnosis for your child. I’m grateful when doctors and healthcare professionals stop to think about it and they don’t just go through the motions- to look at the big picture. It’s helpful when doctors go beyond the appointment to help.  LINKS AND RESOURCES MENTIONED Billy Footwear Kindering Proloquo2Go TUNE INTO THE ONCE UPON A GENE PODCAST Spotify Apple Podcasts Stitcher Overcast CONNECT WITH EFFIE PARKS Website Twitter Instagram

Thu, 22 May 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17214/ https://edoc.ub.uni-muenchen.de/17214/1/Schoen_Simon.pdf Schön, Simon ddc:610, ddc:600, Medizinische F

Der in der Evolution hochkonservierte Wnt-Signalweg spielt in der Embryonalentwicklung, der Stammzellbiologie sowie in der Entstehung von Tumoren eine tragende Rolle. Es werden dabei verschiedene Arten der Wnt-vermittelten Signalübertragung unterschieden: Einerseits der sogenannte kanonische, über β-Catenin vermittelte Wnt-Signalweg und andererseits der Wnt/Ca2+- sowie der planar cell polarity (PCP)-like-Weg, die beide die Signaltransduktion unabhängig von β-Catenin übermitteln. Bislang sind die Mechanismen des Wnt/β-Catenin-Signalweges nur teilweise aufgeklärt, wobei insbesondere das Zusammenspiel von Wnts, Frizzleds und deren Korezeptoren auf funktioneller und struktureller Ebene bis heute nur rudimentär beschrieben wurden. In unserer Arbeitsgruppe konnte in vorangegangenen Arbeiten gezeigt werden, dass der Frizzled-8-Rezeptor (Fzd8) in HT1080-Fibrosarkomzellen im Verhältnis zu den übrigen Fzds sehr stark exprimiert wird und seine erhöhte Expression mit einer Aktivierung des Wnt/β-Catenin-Signalweges sowie mit einer erhöhten Invasivität und Proliferation assoziiert ist (Leitenstern et al., mündliche Mitteilung). Ferner wurde gezeigt, dass die Expression von Fzd8 durch Wnt3a bzw. den kanonischen Wnt-Signalweg negativ reguliert wird (Karow 2008; Kolben, Perobner et al. 2012). Vor diesem Hintergrund sollte nun im Rahmen dieser Arbeit mit Hilfe von Reportergenexperimenten die transkriptionelle Regulation von Fzd8 im Detail untersucht werden. Hierzu wurden die Fzd8-Promotorregion und verschiedene 5’- und 3’-terminale Verkürzungsvarianten in den Vektor pGLuc-Basic kloniert, der eine Quantifizierung der Promotoraktivität mittels des Reporterproteins Gaussia Luciferase erlaubt. Der Fzd8-Promotor konnte durch dieses Verfahren in aktivierende und repressive Elemente unterteilt werden, wobei auch ein putatives Enhancer-Element identifiziert werden konnte. Unter Wnt3a-Stimulation zeigten alle untersuchten Promotorkonstrukte eine verringerte Reporterproteinaktivität, was auf eine negative Regulation von Fzd8 durch Wnt3a hinweist. Interessanterweise aber führten Manipulationen am Wnt/β-Catenin-Signalweg weiter downstream zu entgegengesetzten Ergebnissen. So führte ein APC-Knockdown, der mit einer starken Aktivitätszunahme des Wnt/β-Catenin-Signalweges einhergeht, zu einer Zunahme der Fzd8-Promotoraktivität. Im Gegensatz hierzu ging ein Knockdown von β-Catenin, der einer Inhibierung des Wnt/β-Catenin-Signalweges gleichkommt, mit einer verminderten Fzd8-Promotoraktivität einher. Diese Ergebnisse deuten darauf hin, dass die Wnt3a-vermittelte Repression von Fzd8 auch unabhängig vom kanonischen Wnt/β-Catenin-Signalweg erfolgen könnte. Des Weiteren wurde die Fzd8-Promotorregion einer in silico-Analyse mit der Software MatInspector unterzogen, um mögliche Transkriptionsfaktoren zu identifizieren, die für die Fzd8-Regulation maßgeblich sein könnten. Dabei zeigte sich, dass der Transkriptionsfaktor ZF5, der sowohl aktivierend als auch reprimierend an verschiedenen Promotoren wirken kann, aufgrund des Verteilungsmusters seiner Bindungsstellen am Fzd8-Promotor als möglicher Regulator fungieren könnte. ZF5 konnte dabei als putativ positives Wnt/β-Catenin-Zielgen charakterisiert werden. Es konnte ferner gezeigt werden, dass Fzd8 indirekt über ZF5 durch den Wnt/β-Catenin-Signalweg induziert werden kann. Da ZF5 wie auch Fzd8 in HT1080-Fibrosarkomzellen stark exprimiert wird und die ZF5-Überexpression mit einer Zunahme des Wnt/β-Catenin-Signals einhergeht, könnte hier ein positiver feedback loop vorliegen. Somit könnte diese Amplifikation des Wnt/β-Catenin-Signals von maßgeblicher Bedeutung für die verstärkte Proliferation und Invasivität von mesodermalen Tumoren sein.

Background: Deregulation of Wnt/beta-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein beta-catenin. beta-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto-oncogenes MYC and CCND1 as well as the genes encoding the basic helix-loop-helix (bHLH) proteins ASCL2 and ITF-2B. To identify genes commonly regulated by beta-catenin in colorectal cancer cell lines, we analyzed beta-catenin target gene expression in two non-isogenic cell lines, DLD1 and SW480, using DNA microarrays and compared these genes to beta-catenin target genes published in the PubMed database and DNA microarray data presented in the Gene Expression Omnibus (GEO) database. Results: Treatment of DLD1 and SW480 cells with beta-catenin siRNA resulted in differential expression of 1501 and 2389 genes, respectively. 335 of these genes were regulated in the same direction in both cell lines. Comparison of these data with published beta-catenin target genes for the colon carcinoma cell line LS174T revealed 193 genes that are regulated similarly in all three cell lines. The overlapping gene set includes confirmed beta-catenin target genes like AXIN2, MYC, and ASCL2. We also identified 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are regulated similarly in DLD1 and SW480 cells and one pathway - the steroid biosynthesis pathway - was regulated in all three cell lines. Conclusions: Based on the large number of potential beta-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T cells as well as the large overlap with confirmed beta-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study Wnt/beta-catenin signaling and associated colorectal carcinogenesis. Furthermore, the confirmed and the newly identified potential beta-catenin target genes are useful starting points for further studies.

Mit der intensiven Erforschung von Stammzellen soll zukünftig die Möglichkeit eröffnet werden durch Manipulation von Stammzellpopulationen das regenerative Potential des Organismus zu verstehen und zu nutzen. Darüber hinaus soll mittels genetisch modifizierter Stammzellpopulationen die Grundlagen dafür geschaffen werden schwere Gen- und Immundefekte zu therapieren. Humane mesenchymale Stammzellen (hMSC) versprechen hierbei großes Potential, da sie bereits für systemische Therapieansätze eingesetzt werden. Allerdings sind die theoretischen Grundlagen der Stammzelleigenschaften der hMSC wie Proliferation, Invasion bzw. Migration und die Differenzierungskapazität nur rudimentär verstanden. Aufbauend auf den Ergebnissen von Dr. Marisa Karow und Dr. Thomas Kolben, die eine Beteiligung des Wnt/β-Catenin-Signalweges an der Steuerung dieser Stammzelleigenschaften in ihren Arbeiten nachweisen konnten (Karow 2008; Kolben et al. 2012), sollten in der vorliegenden Arbeit die Rezeption des spezifischen Wnt-Signals und die dabei beteiligten Frizzled (Fzd)-Rezeptoren näher untersucht werden. Da aus den vorigen Arbeiten schon hervorging, dass alle 10 Fzd-Rezeptoren in den hMSC exprimiert werden, sollte über eine qualitative PCR die Expression potentieller Wnt-Liganden sowie der beiden Inhibitoren des Wnt-Weges sFRP1 und WIF1 geprüft werden. Dabei zeigte sich, dass 8 der 19 Wnts sowie der Inhibitor sFRP1 exprimiert werden. Das Wnt-Expressionsmuster der Krebszelllinie HT1080 unterschied sich dagegen nur in der Expression zweier Wnts, was wahrscheinlich auf den mesodermalen Ursprung dieser Krebszelllinie zurückzuführen ist. Im Gegensatz hierzu zeigte sich eine umfangreichere Wnt-Expression in der immortalisierten Zelllinie HEK293, in der 13 der 19 Wnts sowie auch die beiden Inhibitoren sFRP1 und WIF1 nachgewiesen wurden. Für eine detailliertere Aufschlüsselung der Beteiligung einzelner Fzds an der Rezeption eines Wnt-Signals sowie an der basalen Aktivität des Wnt/β-Catenin-Signalweges in hMSC wurde ein TCF/LEF-Reporter-Vektorsystem in die hMSC eingebracht. Als Reportergen kam die sekretierte Form einer Luciferase aus dem Tiefsee-Cephalopoden Gaussia princeps zum Einsatz. Vorteil dieser Luciferase ist, dass die Sekretion in den Überstand eine Evaluierung der Wnt/β-Catenin-Aktivität über die Zeit hinweg erlaubt, ohne die Zellen zerstören zu müssen. Hierbei wurden transient und stabil-transfizierte TCF/LEF-(T-cell-specific transcription factor/lymphoid enhancer-binding factor)-Reporter-hMSC generiert und die Zellkulturbedingungen für nachfolgende Experimente optimiert. Die Evaluierung unterschiedlicher Aktivatoren des Wnt/β-Catenin-Signalweges ergab die stärkste Aktivierung nach Knockdown des Tumorsuppressorgens APC, während ein Knockdown des β-Catenins, dem zentralen Mediator des Wnt-Weges, zu einer signifikanten Reduktion der Gaussia-Luciferase-Aktivität führte. Bei Knockdown-Studien mit den verschiedenen Fzds mittels RNA-Interferenz (RNAi) in den TCF/LEF-Reporter-hMSC zeigte sich, dass Fzd5 und Fzd7 an der Weiterleitung eines Wnt-Signals sowohl unter unstimulierten Bedingungen als auch nach Applikation von Wnt3a beteiligt sind. Weiter zeigte nur noch der Knockdown von Fzd1, dass dieser Rezeptor an der Weiterleitung eines Wnt3a-Signals partizipiert. Um diese Ergebnisse über einen reversen Ansatz in Form einer Überexpression der Fzds zu untersuchen, wurde ein flexibles Klonierungssystem entwickelt, das einen einfachen Austausch von Protein-tags ermöglichen sollte. Nach Bestätigung der Überexpression der Fzds auf mRNA-Ebene und Proteinebene wurde die Wirkung der Überexpression in den TCF/LEF-Reporter-hMSC ohne und mit Wnt3a-Aplikation untersucht. Hier zeigte sich, dass neben der Überexpression von Fzd5 auch Fzd3 und Fzd6 eine Erhöhung der Gaussia-Luciferase-Aktivität und damit eine gesteigerte Aktivität des Wnt/β-Catenin-Signalweges sowohl unter unstimulierten als auch unter stimulierten Bedingungen nach sich zogen. Bei vergleichender Überexpression von Wnt3 oder Wnt3a konnte gezeigt werden, dass Wnt3 – welches hMSC auch endogen exprimieren – sehr viel stärker den Wnt-Weg aktivieren kann als Wnt3a. Um die Auswirkung des kanonischen Wnt-Rezeptors Fzd5 auf die Stammzelleigen-schaften der hMSC zu prüfen, wurde die Expression des Wnt-Zielgens Cyclin D1 und die Proliferation nach Knockdown sowie transienter Überexpression von Fzd5 quantitativ erfasst. Hierbei zeigte sich nach Knockdown von Fzd5 eine signifikante Abnahme der Proliferation, die auch auf Ebene der Wnt-Zielgene mit einer Abnahme der Cyclin D1-Expression einherging. Umgekehrt konnten stabil Fzd5-transfizierte hMSC-Populationen generiert werden, die eine signifikante Steigerung der Cyclin D1-Expression aufwiesen. Zusammenfassend konnte im Rahmen der hier vorliegenden Arbeit gezeigt werden, dass die verschiedenen Fzds in unterschiedlichem Maße an der Weiterleitung eines Wnt-Signals beteiligt sind. Besonders scheinen hier Fzd5 und Fzd7 eine wichtige Rolle in hMSC zu spielen, da sie auch an der Vermittlung eines endogenen Wnt-Signals innerhalb der Stammzellpopulation beteiligt sind. Weiter wurde im Fall von Fzd5 auch die maßgebliche Beteiligung dieses Rezeptors am Erhalt der Proliferationskapazität der hMSC-Population nachgewiesen.

Hintergrund und Zielsetzung: Die aktuelle TNM-Klassifikation berücksichtigt bis dato nicht die tatsächliche Tumorlast bei mehrherdigen Mammakarzinomen. Neuere Daten weisen darauf hin, dass mehrherdige Karzinome im Vergleich zu einherdigen Karzinomen bei identischem TNM-Stadium eine schlechtere Prognose hinsichtlich Gesamtüberleben und Rezidivauftreten haben. Ziel dieser Studie war es, mögliche Unterschiede in der Tumorbiologie von ein- und mehrherdigen Mammakarzinomen zu evaluieren. Material und Methoden: Wir verglichen retrospektiv 57 einherdige mit 55 mehrherdigen Mammakarzinompatientinnen. Aus diesem Gesamtkollektiv isolierten wir eine Matched-Pair-Gruppe mit 46 Patientinnen, deren Kollektive hinsichtlich Tumorgröße, Grading und Lymphknotenstatus übereinstimmten. Die Paraffinschnitte jeder Patientin wurden immunhistochemisch auf die Expression von E-Cadherin, Beta Catenin und Mucin-1 untersucht. Ergebnisse: Die E-Cadherin-Expression war bei den mehrherdigen Mammakarzinomen signifikant reduziert gegenüber den einherdigen Karzinomen (Gesamtkollektiv: p

Thu, 7 Mar 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15583/ https://edoc.ub.uni-muenchen.de/15583/7/Lechner_Felix.pdf Lechner, Felix

Thu, 21 Feb 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15427/ https://edoc.ub.uni-muenchen.de/15427/1/Stroehle_Bernadette_Sarah.pdf Ströhle, Bernadette Sarah ddc:610, ddc:60

Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating β-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/β-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target. Similar to patient tumor specimen the H295 cell line derived from an ACC harbors a natural activating β-catenin mutation. We herein assess the in vitro and in vivo effect of β-catenin inactivation using a doxycyclin (dox) inducible shRNA plasmid in H295R adrenocortical cancer cells line (clone named shβ). Following dox treatment a profound reduction in β-catenin expression was detectable in shβ clones in comparison to control clones (Ctr). Accordingly, we observed a decrease in Wnt/βcatenin-dependent luciferase reporter activity as well as a decreased expression of AXIN2 representing an endogenous β-catenin target gene. Concomitantly, β-catenin silencing resulted in a decreased cell proliferation, cell cycle alterations with cell accumulation in the G1 phase and increased apoptosis in vitro. In vivo, on established tumor xenografts in athymic nude mice, 9 days of β-catenin silencing resulted in a significant reduction of CTNNB1 and AXIN2 expression. Moreover, continous β-catenin silencing, starting 3 days after tumor cell inoculation, was associated with a complete absence of tumor growth in the shβ group while tumors were present in all animals of the control group. In summary, these experiments provide evidences that Wnt/β-catenin pathway inhibition in ACC is a promising therapeutic target.

Humane mesenchymale Stammzellen (hMSC) haben in den vergangenen Jahren auf-grund ihres potenziellen Einsatzes in der regenerativen Medizin sowie in der Prävention und Behandlung diverser Krankheiten ein großes wissenschaftliches Interesse geweckt. Einen wichtigen Aspekt stellt in diesem Zusammenhang die Regulation von Stammzell-funktionen durch Signalwege wie beispielsweise den Wnt/β-Catenin-Signaltransduk-tionsweg dar. Während am Signalweg beteiligte Komponenten und Teilfunktionen bereits beschrieben sind, existieren bezüglich der Initiation der Signaltransduktion an der Zelloberfläche auf Rezeptorebene lediglich rudimentäre Kenntnisse. Vor diesem Hintergrund wurde in dieser Arbeit die molekulare Funktion der Wnt-Ko-rezeptoren LRP5 und LRP6 (low-density lipoprotein receptor-related protein) im Wnt/β-Catenin-Signalweg von hMSC genauer untersucht. Für die spezifische Quantifizierung β-Catenin-abhängiger Transkriptionsprozesse wurde zunächst ein TCF/LEF-Reportergen-System in hMSC etabliert. In diesem System erfolgt die Expression des Reporterproteins erst nach Translokation von β-Catenin in den Zellkern und dessen Assoziation mit Transkriptionsfaktoren der TCF/LEF-Familie. Im Vergleich mit dem konventionellen TOP/FOP-Flash-Reportergen-System zeigte das TCF/LEF-Reportergen-System eine deutlich höhere Sensitivität. Mittels vergleichender Studien zur molekularen Funktion von LRP5 und LRP6, die neben der RNA-Interferenz (RNAi)-basierten Technologie auch Überexpressionsstudien und Rescue-Experimente beinhalteten, konnte eindeutig gezeigt werden, dass LRP6 eine entscheidende Rolle in der β-Catenin-vermittelten Signaltransduktion von hMSC übernimmt. Nach Applikation von Wnt-3a führte RNAi gegen LRP6 zu einer starken Ab-nahme der Wnt/β-Catenin-Signaltransduktion, wohingegen der Knockdown von LRP5 keine Veränderung zeigte. In einem umgekehrten Ansatz resultierte die Überexpression von LRP6 in einer starken Aktivierung des Wnt/β-Catenin-Weges, während die Über-expression von LRP5 keinen nachhaltigen Einfluss zeigte. Darüber hinaus führte in LRP6 -Knockdown-hMSC die Überexpression von LRP6 – jedoch nicht die von LRP5 – zur Rekonstitution der Wnt-3a-induzierten, β-Catenin-vermittelten Signaltransduktion. Diese Daten weisen LRP6 als den Hauptrezeptor für die Wnt-3a/β-Catenin-vermittelte Signaltransduktion in hMSC aus, wobei diese Funktion nicht durch LRP5 ersetzt werden kann. Da der Wnt/β-Catenin-Signalweg eng mit Differenzierungsprozessen assoziiert ist, wurde in diesem Kontext die Bedeutung der Wnt-Korezeptoren in hMSC evaluiert. Nach Knockdown von LRP6 war eine Differenzierung in die adipogene Richtung zu beobachten, die mit der Bildung fettähnlicher Vakuolen und einer erhöhten Expression des Transkriptionsfaktors PPAR-γ (Peroxisom-Proliferator-aktivierter Rezeptor-γ) assoziiert war. Unter dem Einsatz adipogener Zusätze konnte die Differenzierung von LRP6-Knockdown-hMSC in fettähnliche Zellen weiter verstärkt werden, was mit einer deutlich gesteigerten Akkumulation von Fettvakuolen sowie einer weiteren Erhöhung der PPAR-γ Expression einherging. Interessanterweise resultierte die Überexpression von LRP6 in diesen fettähnlichen Zellen in einer Zunahme der Wnt/β-Catenin-Signaltransduktion mit einer gleichzeitigen Abnahme der Expression von PPAR-γ. Zusammenfassend zeigen diese Erkenntnisse, dass LRP6 nicht nur in der Wnt-3a-induzierten, β-Catenin-vermittelten Signaltransduktion von hMSC eine tragende Rolle spielt, sondern auch für die Suppression der Differenzierung von hMSC in die adipogene Linie und damit für die Aufrechterhaltung des Stammzellcharakters entscheidend ist. Somit stellt der Wnt-Korezeptor LRP6 ein vielversprechendes Ziel zur therapeutischen Manipulation von hMSC in zukünftigen klinischen Anwendungen, wie z.B. der regenerativen und präventiven Medizin, dar.

Wnt/β-catenin signaling is of fundamental importance in the regulation of self-renewal, migration/invasion, and differentiation of human mesenchymal stem cells (hMSCs). Because little information is available about the function of Frizzled receptors (Fzds) as the main receptors of Wnt proteins in hMSCs, we first performed comparative Fzd mRNA expression profiling. Fzd9 and Fzd10 were not expressed in hMSCs. While Fzd3 was expressed at low levels in hMSCs, the other Fzds exhibited high expression rates. Activation and repression of Wnt signaling in hMSCs revealed that the expression levels of Fzd1, Fzd6, and Fzd7 are positively correlated with the Wnt/β-catenin activation status, whereas Fzd8 exhibited an inverse relation. For studying the functional relevance of Fzds in Wnt/β-catenin signaling, RNA interference, ectopic expression studies, and rescue approaches were performed in hMSCs carrying a highly sensitive TCF/LEF reporter gene system (Gaussia luciferase). We found that, Fzd1, Fzd5, Fzd7, and Fzd8 are largely involved in Wnt/β-catenin signaling of hMSCs. Moreover, the knockdown of Fzd5 can be compensated by the ectopic expression of Fzd7. Conversely, the ectopic expression of Fzd5 in Fzd7-knockdown hMSCs resulted in a rescue of Wnt/β-catenin signaling, pointing to a functional redundancy of Fzd5 and Fzd7.

Thu, 30 Jun 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13188/ https://edoc.ub.uni-muenchen.de/13188/1/Kolben_Thomas.pdf Kolben, Thomas

Fri, 11 Feb 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12685/ https://edoc.ub.uni-muenchen.de/12685/1/Scheel_Silvio_K.pdf Scheel, Silvio ddc:570, ddc:500, Fakultät für Biologie

Background: Genetically, colorectal cancers (CRCs) can be subdivided into tumors with chromosomal instability (CIN) or microsatellite instability (MSI). In both types of CRCs genes that are involved in the degradation of beta-CATENIN are frequently mutated. Whereas in CIN CRCs APC (Adenomatous Polyposis Coli) is affected in most cases, high grade MSI (MSI-H) CRCs frequently display mutations in various genes, like the APC-, AXIN2- or CTNNBI (beta-CATENIN) gene itself. Recently in Wilms tumors, WTX (Wilms tumor gene on the X-chromosome) was discovered as another gene involved in the destruction of beta-CATENIN. As the WTX-gene harbors a short T(6)-microsatellite in its N-terminal coding region, we hypothesized that frameshift-mutations might occur in MSI-H CRCs in the WTX gene, thus additionally contributing to the stabilization of beta-CATENIN in human CRCs. Methods: DNA was extracted from 632 formalin-fixed, paraffin-embedded metastatic CRCs (UICCIV) and analyzed for MSI-H by investigating the stability of the highly sensitive microsatellite markers BAT25 and BAT26 applying fluorescence capillary electrophoresis (FCE). Then, in the MSI-H cases, well described mutational hot spot regions from the APC-, AXIN2- and CTNNBI genes were analyzed for genomic alterations by didesoxy-sequencing while the WTX T(6)-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T(5)-repeats were subcloned and mutations were validated using didesoxy-sequencing. Furthermore, the KRAS and the BRAF proto-oncogenes were analyzed for the most common activating mutations applying pyro-sequencing. mRNA expression of WTX from MSI-H and MSS cases and a panel of colorectal cancer cell lines was investigated using reverse transcription (RT-) PCR and FCE. Results: In our cohort of 632 metastatic CRCs (UICCIV) we identified 41 MSI-H cases (6.5%). Two of the 41 MSI-H cases (4.8%) displayed a frameshift mutation in the T(6)-repeat resulting in a T(5) sequence. Only one case, a male patient, expressed the mutated WTX gene while being wild type for all other investigated genes. Conclusion: Mutations in the WTX-gene might compromise the function of the beta-CATENIN destruction complex in only a small fraction of MSI-H CRCs thus contributing to the process of carcinogenesis.

Background: Most colorectal carcinomas are driven by an activation of the canonical Wnt signalling pathway, which promotes the expression of multiple target genes mediating proliferation inavasion and invasion. Upon activation of the Wnt signalling pathway its key player beta-catenin translocates from the cytoplasm to the nucleus and binds to members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF-1) family namely LEF-1 and TCF4 which are central mediators of transcription. In this study we investigated the expression of beta-Catenin, LEF1 and TCF4 in colorectal carcinomas and their prognostic significance. Methods: Immunohistochemical analyses of LEF-1, TCF4 and nuclear beta-Catenin were done using a tissue microarray with 214 colorectal cancer specimens. The expression patterns were compared with each other and the results were correlated with clinicopathologic variables and overall survival in univariate and multivariate analysis. Results: LEF-1 expression was found in 56 (26%) and TCF4 expression in 99 (46%) of colorectal carcinomas and both were heterogenously distributed throughout the tumours. Comparing LEF-1, TCF4 and b-catenin expression patterns we found no correlation. In univariate analysis, TCF4 expression turned out to be a negative prognostic factor being associated with shorter overall survival (p = 0.020), whereas LEF-1 expression as well as a LEF-1/TCF4 ratio were positive prognostic factors and correlated with longer overall survival (p = 0.015 respectively p = 0.001). In multivariate analysis, LEF-1 and TCF4 expression were confirmed to be independent predictors of longer respectively shorter overall survival, when considered together with tumour stage, gender and age (risk ratio for LEF-1: 2.66; p = 0.027 risk ratio for TCF4: 2.18; p = 0.014). Conclusions: This study demonstrates different prognostic values of LEF-1 and TCF4 expression in colorectal cancer patients indicating different regulation of these transcription mediators during tumour progression. Moreover both factors may serve as new potential predictive markers in low stage colon cancer cases in advance.

Mesenchymale Stammzellen (MSC) stellen aufgrund ihres Differenzierungspotentials einen großen Hoffnungsträger in der regenerativen Medizin dar. Entsprechend zahlreicher zell- und tierexperi-menteller Untersuchungen scheint die klinische Anwendung dieser adulten Stammzellpopulation im Rahmen einer Zelltherapie in greifbare Nähe zu rücken, wobei MSC als Basis für einen Patien-ten-spezifischen Zell- und Gewebeersatz dienen könnten. In welcher Weise die regenerative Kapazität der MSC durch spezielle Signaltransduktionsmechanismen gesteuert wird, ist jedoch noch weitgehend unbekannt. Vor diesem Hintergrund wurde in der hier vorliegenden Arbeit der Wnt/β-Catenin-Signaltrans-duktionsweg sowohl in humanen (hMSC) als auch in murinen (mMSC) mesenchymalen Stamm-zellen untersucht. Diesem komparativen Ansatz lag das Ziel zugrunde, Gemeinsamkeiten und Unterschiede in diesen beiden Zellentitäten zu evaluieren, um damit langfristig den Grundstein für die Übertragbarkeit von Daten aus nachfolgend geplanten murinen in vivo-Modellen auf die klinische Situation legen zu können. Hierzu wurden zunächst die Basis-Komponenten des Wnt/β-Catenin-Signalweges vergleichend analysiert. Eine Aktivierung des Wnt-Signalweges wurde über Stimulation mit Wnt3a bzw. LiCl in beiden Zellspezies sowie in einem RNA-Interferenz (RNAi)-basierten Ansatz durch Knockdown der für den β-Catenin-Abbaukomplex essentiellen Proteine APC und Axin2 in hMSC erreicht, während eine Inhibtion durch die Transfektion von small interfering RNAs (siRNAs) gegen das transkrip-tionsaktivierende Protein β-Catenin bzw. den Wnt-Korezeptor LRP5 induziert wurde. Dabei zeigten sich neben zahlreichen Gemeinsamkeiten unter anderem hinsichtlich der Proliferation auch klare Unterschiede zwischen hMSC und mMSC. Dies betraf insbesondere die Steuerung von Matrix-Metalloproteinase (MMP)-mediierten Invasionsprozessen, die im Falle von hMSC eine deutliche Wnt-Abhängigkeit aufwiesen, während die Invasionsfähigkeit von mMSC nicht durch den Wnt-Signalweg reguliert wurde. Diese Unterschiede in den zellulären Phänotypen spiegelten sich vorwiegend in einer Spezies-divergenten Regulation der Matrix-Metalloproteinase MT1-MMP wider, da nur in hMSC die Aktivierung der Wnt-Signaltransduktionskaskade mit einer vermehrten MT1-MMP-Expression einherging. Darüber hinaus konnte das Tcf/Lef-Reporter-System in mMSC etabliert werden, das die Quanti-fizierung β-Catenin-abhängiger Expression ermöglicht. Dies erfolgt mit Hilfe eines Reporter-proteins, dessen Expression nur nach Translokation von β-Catenin in den Zellkern induziert wird. Mit diesem System konnte unter anderem auch der Nachweis der funktionellen Plasmid-kodierten Wnt3a-Expression erbracht werden. Derartig generierte Reporter-mMSC könnten vor allen Dingen hinsichtlich einer Anwendung im in vivo-Mausmodell von großem Vorteil sein, da Wnt-aktive MSC mittels eines in vivo-Imaging-Systems visualisiert werden können, um ihre Rolle bei Geweberegenerationsprozessen aufzuklären. In einem weiteren Teilprojekt wurde die Wirkung von Dkk-1, einem Inhibitor des kanonischen Wnt-Signalweges, in hMSC eingehend untersucht. Dabei stand die Analyse der Wechselwirkungen zwischen Dkk-1 und seinem Rezeptor LRP6 im Vordergrund. Versuche zum LRP6-Knockdown brachten ein komplexes Regulationssystem zutage, das eine feinjustierte Balance zwischen akti-vierenden und inhibierenden Signalen impliziert. Die Ergebnisse zusätzlicher RNAi-basierter Experimente wiesen außerdem auf eine funktionelle Divergenz von LRP5 und LRP6 hin. So vermittelt der Wnt-Korezeptor LRP5 vornehmlich aktivierende Signale, wie sie z.B. durch Wnt3a ausgelöst werden, während LRP6 hauptsächlich eine repressive Funktion beispielsweise durch Bindung von Dkk-1 zuzuordnen ist. Da neben den LRP-Rezeptoren auch Frizzled-Rezeptoren (Fzd) eine wesentliche Rolle bei der Wnt-Signalerfassung spielen, wurde zunächst das Fzd-Expressionsprofil in hMSC und mMSC mittels semiquantitativer RT-PCR-Analysen näher untersucht. Dabei zeigte sich, dass alle bisher bekannten 10 Fzds auch in MSC exprimiert werden, dieses jedoch in unterschiedlichem Ausmaß. Zudem ergaben Wnt3a-Stimulationsexperimente in hMSC, dass die Expression von Fzd8 negativ durch Wnt3a beeinflusst wird. Um die Bedeutung von Fzd8 näher zu evaluieren, wurden daher Fzd8-Knockdown-Experimente durchgeführt. Diese ließen erkennen, dass die hMSC-Proliferation maß-geblich von der Fzd8-Expression abhängt, wobei allerdings Fzd8 keinen direkten Rezeptor für Wnt3a darstellt. Zusammenfassend spiegeln die in der vorliegenden Promotionsarbeit erhobenen Daten zum Teil eindeutige Unterschiede zwischen basalen Wnt-regulierten Prozessen in hMSC und mMSC wider, denen insbesondere bei der präklinischen Validierung von therapeutischen Strategien in Maus-modellen eine tragende Rolle zukommt. Da der Wnt/β-Catenin-Signalweg maßgeblich an der Steuerung des invasiven Verhaltens von hMSC beteiligt ist, wie dies in ähnlicher Weise von anderen Forschergruppen auch für die Metastasierung von Tumorzellen nachgewiesen werden konnte, erscheint es zukünftig von vorrangigem Interesse, die hier erhobenen in vitro-Daten in einem in vivo-Mausmodell zu evaluieren. In diesem Kontext kann allerdings nur durch einen komparativen Ansatz, wie er dieser Arbeit zugrunde liegt, die Basis für ein Spezies-relevantes drug design bezüglich des Wnt-Signalweges entwickelt werden, um schließlich aussagekräftige Stamm-zelltherapien bzw. Anti-Tumorstrategien entwickeln zu können.

Ziel der Arbeit war die Etablierung RNA Interferenz basierender Systeme zur Senkung der Proteinspiegel von b- und g-Catenin und die darauf aufbauende Evaluierung potentieller b-Catenin Zielgene mittels Microarray Technologie. Der Schwerpunkt sollte dabei auf die Untersuchung von im Wnt-Signalweg beteiligten Genen gelegt werden. Wir konnten in den kolorektalen Karzinomzelllinien SW-480, DLD-1 und HT-29 mehrere voneinander unabhängige siRNA sowohl gegen b- als auch g-Catenin etablieren. Dabei stand neben ausreichender Senkung der Protein- und mRNA-Spiegel auch die Vermeidung unerwünschter Effekte auf die Zellen im Mittelpunkt dieser Etablierungsarbeit. Im Rahmen der vorliegenden Arbeit konnten vier neue funktionale Zelllinien etabliert werden. Sowohl in HT-29 als auch in SW-480 konnte ein universell einsetzbarer TET-Repressor stabil integriert werden. Die Linien HT-29TR und SW-480TR können als Basis für induzierbare Expressionssysteme verwendet werden. Auf Grundlage der Linie SW-480TR konnten Zelllinien etabliert werden, die durch Doxycyclin vermittelte Induktion shRNA gegen b-Catenin exprimieren. Mittels dieser Linien können Effekte, die durch die Senkung der b-Catenin Proteinspiegel hervorgerufen werden, einfach, schnell und zuverlässig in einem breiten Spektrum an in vitro und in vivo Assays untersucht werden. Im Rahmen einer Microarray Analyse des Transkriptoms von SW-480 Zellen nach RNA Interferenz basierter Senkung der b-Catenin Proteinspiegel wurde DKK4 als sehr stark reguliertes Gen auffällig. Mittels RT-PCR konnten wir bestätigen, dass die Expression von DKK4 direkt von den vorliegenden b-Catenin Proteinspiegeln abhängig ist. Basierend auf einer in silico Analyse des DKK4 Promoters wurden mit Hilfe von Reportergen Konstrukten der für die Aktivierbarkeit durch b-Catenin verantwortliche Abschnitt des DKK4 Promoters bestimmt. Wir konnten darstellen, dass die Aktivierung des DKK4 Promotors sowohl von der Präsenz von b-Catenin als auch von TCF4 abhängt. Die Expression von DKK4 bedingt die Gegenwart des aus TCF4 und b-Catenin bestehenden Transaktivierungskomplexes, der für die Aktivierung Wnt-abhängiger Transkription verantwortlich zeichnet. Gleichzeitig stellt der DKK4 Promoter durch die Präsenz von TCF-Bindestellen eine Zielstruktur für diesen Transaktivierungskomplex dar. Unsere Daten belegen, dass DKK4 ein b-Catenin Zielgen darstellt. Wir konnten auflerdem zeigen, dass die Aktivierung Wnt-abhängiger Transkription durch Gabe von rekombinantem DKK4 gehemmt werden kann. Es konnte im Rahmen dieser Arbeit dargestellt werden, dass die Expression des Wnt-Antagonisten DKK4 in einen negativen Feedback Mechanismus, der durch b-Catenin autoreguliert wird, eingebunden ist. Der vermutete Feedback Mechanismus stellt sich wie folgt dar. Wnt-Faktoren aktivieren die Transkription b-Catenin abhangiger Zielgene, darunter auch DKK4. DKK4 initiiert durch Bindung an LRP und Kremen den Abbau von b-Catenin und verhindert somit die weiterführende Transkription von Wnt-b-Catenin Zielgenen. DKK4 übernimmt als autokriner Inhibitor des Wnt-Signalwegs feinregulatorische Aufgaben, um bei moglicher Bedarfsdeckung b-Catenin abhängiger Gentranskription die weitere Aktivierung durch Wnt-Faktoren zu verhindern. Fur den Fall, dass DKK4 als parakriner Inhibitor agiert, wäre es denkbar, dass DKK4 von Zellen im unteren Teil der Krypte, die in hohem Maße durch Wnt-Faktoren aktiviert, werden sezerniert wird, um die darüber liegenden Zellen durch Abschirmung von weiteren Wnt-Signalen vor überschieflender Expression Wnt-b-Catenin abhängiger Gene und Proliferation zu schutzen und die Differenzierung dieser Zellen einzuleiten. Die unkontrollierte Expression von Wnt-b-Catenin Zielgenen nimmt bei der Entstehung kolorektaler Karzinome eine entscheidende Schlüsselposition ein. Zur Etablierung neuer Diagnostik- und Therapieoptionen bedarf es daher auch der stetigen Erweiterung des Verständnisses des Wnt-Signalwegs. Diese Arbeit fügt ein weiteres Rädchen in die komplexe Mechanik dieses Signalwegs ein.

Die IGFBP-2-Serumkonzentrationen sind beim menschlichen Kolonkarzinom mit dem Tumorstadium signifikant positiv korreliert und sogar als prognostischer Marker für eine Wiedererkrankung nach Tumorresektion informativ. Dennoch ist die spezifische Rolle von IGFBP-2 für die Entstehung und Progression des Kolonkarzinoms völlig unklar. In vitro wurden für IGFBP-2 sowohl positive als auch negative Effekte auf das Wachstum normaler und maligner Zellen nachgewiesen. Für die zweifelsfreie Zuordnung spezifischer Effekte in vivo wurde in der vorliegenden Arbeit das IGFBP-2-transgene Mausmodell verwendet. In diesem Modell wurde die Kolonkarzinogenese chemisch induziert. Um herauszufinden, in welchem Stadium der mehrstufigen Kolonkarzinogenese IGFBP-2 von Bedeutung ist, wurden die Analysen 10 und 34 Wochen nach Beginn der Behandlung mit dem Karzinogen durchgeführt. Zum früheren Analysezeitpunkt wurde festgestellt, dass IGFBP-2 zunächst die Entstehung von kleinen hyperplastischen aberranten Krypten Foci (ACF) förderte, sich diese ACF aber in der späteren Phase zurückentwickelten und keinen Einfluss auf die Tumorprävalenz hatten. Im Vergleich zum Wildtyp entwickelten die IGFBP-2-transgenen Mäuse jedoch weniger dysplastische ACF. Dysplastische ACF in IGFBP-2-transgenen Mäusen bildeten kleinere Foci und wiesen einen deutlich geringeren Dysplasiegrad auf. Größere ACF des transgenen Genotyps stellten den hyperplastischen ACF-Typ dar. Interessanterweise war die Expression von β-Catenin in den ACF transgener Tiere gegenüber dem Wildtyp deutlich reduziert. Diese Ergebnisse deuten darauf hin, dass IGFBP-2 einen hemmenden Effekt bereits im frühen Stadium der Kolonkarzinogenese ausübt. Zum späteren Untersuchungszeitpunkt unterschied sich die Tumorprävalenz zwischen den beiden Genotypen nicht voneinander. Jedoch war das Volumen der Adenome der IGFBP-2-transgenen Gruppe um das 2,3-fache kleiner als beim Wildtyp, was durch einen geringeren Anteil an proliferierenden Tumorzellen bedingt war und sich bereits bei den ACF der frühen Phase abzeichnete. Zudem wurde, in Übereinstimmung mit den Ergebnissen der früheren Phase, eine deutlich geringere nukleäre Akkumulation von β-Catenin in den Adenomen IGFBP-2-transgener Tiere beobachtet. Diese Ergebnisse zeigen, dass IGFBP-2 sowohl in der frühen als auch in der späteren Phase der Kolonkarzinogenese einen hemmenden Effekt auf das Wachstum von dsyplastischen ACF und Tumoren hat, indem es die Tumorlast reduziert und die Akkumulation von nukleärem β-Catenin inhibiert. Um Effekte von IGFBP-2 auch auf Ebene der Genexpression zu berücksichtigen, wurde eine Expressionsanalyse von Kandidatengenen, die aus der Literatur im Zusammenhang mit einer Überexpression von IGFBP-2 bekannt waren, durchgeführt. Interessanterweise ergab die Real-Time PCR Analyse eine erhöhte MMP2-, TIMP1- und NFκB-mRNA Expression in Tumoren, nicht aber im normalen Kolongewebe von IGFBP-2-transgenen Mäusen. Aus der konditionalen Überexpression von Invasions- und Migrations-assoziierten Genen im Tumor von IGFBP-2-transgenen Mäusen könnte auf einen möglichen Effekt von IGFBP-2 auf die Metastasierung geschlossen werden. Die Relevanz dieser veränderten Genexpression sollte in einem Metastase-Modell weiterführend untersucht werden.

Kim-1 (kidney injury molecule-1), a member of the TIM (T-cell immunglobulin mucin) family is a glykosylated type 1 transmembrane protein, which is markley upregulated during the repair of renal epithel cells after ischemia and in malignant and cystic kidney disease, while expressed at low levels in normal kidneys. In previous experiments Kim-1 has been shown to localize in primary cilia of cultured renal epithelial cells in-vitro. Primary cilia have been shown to play a role in development and recently cilial dysfunction has been shown to be the unifying defect causing polycystic kidney disease. Kim-1 interacts with Inversin, a ciliary protein, which is mutated in Type 2 nephronophthisis, and which has been shown to modulate the Wnt pathway by targeting Dishevelled (Dvl) for proteasomal degradation. The expression of Wnt proteins is necessary for many fundamental processes in differentiation, proliferation, polarity and adhesion. Wnts play an important role in the early embryonic development. There is only limited knowledge on the physiological role of Kim-1 during differentiation, proliferation and survival of tubular epithel cells after renal injury. The aim of this study was to characterize the role of Kim-1 in epithelial polarization and ciliogenesis of tubular epithel cells outside of pathological injury models. Additionally the role of Kim-1 in the Wnt signaling pathway was examined. Renal tubular cells were analyzed phenotypically with immunfluorescence staining. Protein expression levels were detected by SDS PAGE and Western blot. Signaltransduction activity was measured in luciferase reporter assays. Protein overexpression was achieved with transient transfection of plasmid DNA in cultured cells after calcium chloride precipitation. Gene reduction was achieved through lentivirus-mediated transduction of short interfering RNAs. Examination of immunfluorescence stained cryosections showed, that Kim-1 is localized in-vivo in primary cilia of tubular epithelial cells in mouse kidneys. Kim-1 expression levels correlate with the polarization of tubular epithel cells in-vitro. Reporter assays using TOP FLASH luciferase constructs revealed that Kim-1 reduced the Dvl-induced TOP FLASH activation and decreased steady-state levels of ß-Catenin in HEK 293t cells, suggesting that Kim-1 antagonizes the canonical Wnt pathway. Conversely Kim-1 expression increased AP-1 activity, which has been linked to the non-canonical Wnt pathway. Phenotypical characterization of two different immortalized renal epithelial cell lines after Kim-1 knock-down showed no changes in epithelial polarization, or ciliogenesis. These results suggest that Kim-1 is a ciliary protein that probably plays no active role in cell polarization or ciliogenesis, but may participate in the Wnt signaling pathway. Kim-1 may act as a molecular switch between the canonical and non-canonical Wnt pathway and thus may play a role in epithelial differentiation.

Fri, 23 Sep 2005 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/4238/ https://edoc.ub.uni-muenchen.de/4238/1/Dolce_Luca.pdf Dolce, Luca ddc:570, ddc:500, Fakultät für B

BRG1 is a conserved subunit of the SWI/SNF family of ATP dependent chromatin remodeling complexes. These complexes play an important role in the transcription of various genes by making promoters accessible to the transcription machinery. Mutations in BRG1 have been connected to various cancers. In addition, a BRG1 knock-out in mice is lethal at the periimplantation stage, while BRG1 heterozygote mice are predisposed to exencephaly and tumors of epithelial origin, showing the importance of BRG1 in normal development and disease. In this study, I used Xenopus laevis to study the role of BRG1 because this system allows manipulation of endogenous protein levels by the use of antisense oligonucleotide mediated knock-down as well as interference analysis at early stages of development by overexpression of wild type and dominant negative protein variants. Since BRG1 is conserved among all vertebrates, I initially studied the role of BRG1 in Xenopus development by overexpression of wild type and dominant negative human BRG1. Overexpression of dominant negative human BRG1 gave a ventralized phenotype suggesting a role of BRG1 in dorsal-ventral patterning. The specificity of phenotypes was confirmed by using wild type human BRG1. On the other hand, overexpression of wild type and dominant negative variants of human BRM showed no developmental phenotypes. Prompted by these results, a frog brg1 cDNA was cloned by searching the Xenopus laevis EST database, using human BRG1 as a query. In addition, monoclonal antibodies specific to xBRG1 were raised and characterized. The expression pattern of Xbrg1 was found to be ubiquitous until gastrula stage and is tissue specific from neurula stage onwards. A Xenopus homologue of INI1, a subunit of SWI/SNF chromatin-remodeling complex, was cloned using database search. The expression pattern of Xini1 was found to be similar to Xbrg1. Using site directed mutagenesis, a dominant negative construct of xBRG1 was made by mutating the conserved lysine into arginine (K770R). Loss and gain of function studies showed that BRG1 is involved in AP axis formation during Xenopus development. The gain of function studies were done by overex-pressing wild type and dominant negative xBRG1, while loss of function studies were done using highly specific antisense morpholino oligos. Specificity of morpholino treatment was further proven by the rescue of ventralized phenotypes of morphant embryos by overexpression of human BRG1. It was found that BRG1 knock-down affects several tissues as assessed by in-situ hybridization using tissue specific markers. To determine the molecular explanation for these pleiotropic effects, several genes involved in early patterning of Xenopus embryo during organizer formation were analyzed. The analysis was done using whole mount in-situ hybridization, revealing the spatial gene expression pattern. This analysis revealed that BRG1 mostly affects WNT signaling dependent genes required for dorsal mesoderm formation while leaving pan-mesodermal genes unaffected. Furthermore the genetic interaction of BRG1 with the WNT pathway was confirmed by epistasis experiments showing that overexpression of β-CATENIN can rescue the xBrg1 antisense morpholino oligos dependent ventralized phenotypes as well as formation of secondary axis by overexpression of β-CATENIN could be prevented by BRG1 knock-down. Since the whole embryo represents a complex situation whereby many signaling pathways interact with each other and influence the outcome, the animal cap system was used to analyze the effect of BRG1 on various signaling pathways by analyzing corresponding direct target genes. Animal cap assays showed that the effect of BRG1 is signal specific. Moreover, among the affected signaling pathways, BRG1 knock-down affected only specific genes. These results showed that the BRG1 effect is gene and signal specific. The importance of WNT signaling has also been shown in cancer as well as in haematopoietic and embryonic stem cell self renewal. Given the importance of the WNT signaling, the role of BRG1 on the WNT signaling pathway was further investigated. Treatment of animal cap cells with various doses of Wnt8 mRNA showed the differential requirement of the WNT signal for maximal stimulation of direct target genes. The direct target genes of the WNT pathway showed various degrees of reduction in their maximal stimulation upon BRG1 protein knock-down. The requirement of BRG1 for proper stimulation of the WNT target genes was further confirmed by overexpression of xBRG1 under sub-optimal conditions of WNT stimulation. A major conclusion from these experiments is that BRG1 protein defines signaling thresholds for WNT-mediated activation of target genes. This implies that chromatin remodeling complexes are part of the machinery, which translates inductive signals into spatial gene expression domains.

Das kolorektale Karzinom (CRC) ist weltweit der zweithäufigste bösartige Tumor. Fernmetastasen eines CRC treten zumeist in Leber und Lunge auf, sind nur in seltenen Fällen operativ entfernbar und sprechen nicht auf derzeit verfügbare Chemotherapien an. Die Identifizierung von Genen und die Charakterisierung der molekularen Mechanismen, durch welche sie zur Tumorprogression eines CRC beitragen, liefert eine Grundlage für die Prävention oder Behandlung der zumeist tödlich verlaufenden Erkrankung. Das Ziel dieser Arbeit war die Identifizierung von Genen, die für die Entwicklung des metastatischen Phänotyps in Kolonkarzinomzellen verantwortlich sind. Hierzu wurden mittels der Genchip-Technologie die Transkriptionsprofile von insgesamt fünf Tumor-Zelllinien erstellt und nach stringenten Auswahlkriterien miteinander verglichen. Zuerst wurde ein CRC-Zellmodell verwendet, das aus einer in der Nacktmaus nicht-metastasierenden humanen Primärtumor-Zelllinie KM12C und zwei davon abgeleiteten, stark zur Leber metastasierenden Zelllinien, KM12SM und KM12L4A bestand. In diesem überwiegend isogenen Zellmodell wurden 145 Gene in beiden metastasierenden Zelllinien als dereguliert im Vergleich zu der nicht-metastasierenden Zelllinie identifiziert. Die Transkriptionsprofile eines weiteren humanen CRC-Zellmodells, bestehend aus einer nicht-metastasierenden Zelllinie HCT116 und einer stark zur Lunge metastasierenden Zelllinie HCT116U5.5 wurden ebenfalls verglichen und 72 Gene als differentiell exprimiert identifiziert. Ein Vergleich der Datensätze aller Transkriptionsprofile ermöglichte die Identifizierung von Vimentin und Trop-2 (TACSTD2, „Tumorassoziierter Vermittler von Kalziumsignalen“) als in allen metastasierenden Zelllinien überexprimiert. Die hohe Homologie des bisher noch weitestgehend unerforschten Trop-2 zu Trop-1 (Ep-CAM), einem regulatorischen Zell-Zell-Adhäsionsmolekül, das überwiegend von aggressiven Tumorarten exprimiert wird und die Proliferation von Tumorzellen stimulieren kann, machten Trop-2 für weiterführende Expressions- und Funktionsstudien zu einem interessanten Zielgen. Im Rahmen dieser Arbeit durchgeführte Expressionsstudien in einer Vielzahl von Tumorbiopsien zeigten deutlich erhöhte mRNA- und Proteinspiegel von Trop-2 in Metastasen aus CRC im Vergleich zu den Primärtumorgeweben. Zusätzlich zu diesem neuen Befund in CRC wurde in dieser Arbeit erstmalig eine erhöhte Expression von Trop-2 in Schild-drüsenkarzinomen und in nicht-kleinzelligen Lungenkarzinomen im Vergleich zu den korrespondierenden Normalgeweben nachgewiesen. Zur Untersuchung der funktionellen Bedeutung von Trop-2 für die Metastasierung von Karzinomzellen des Kolons wurden stabile Transfektanten für Trop-2 in der nicht-metastasierenden Zelllinie KM12C generiert. Die konstitutiv Trop-2 exprimierenden Zellklone zeigten erhöhte Cyclin D1-Proteinspiegel bei unveränderter Zellproliferation gegenüber den Vektorkontrollen. Die Induktion von Cyclin D1 könnte mit der Identifizierung eines ebenfalls erhöhten Proteinspiegels von ß-Catenin in den Trop-2 exprimierenden Transfektanten in Zusammenhang stehen. Somit liefert diese Arbeit erste Hinweise auf einen möglichen Einfluss von Trop-2 auf den Tcf/ß-Catenin Signalweg. Zusätzlich demonstrierte ein Migrationstest erhöhte mobile Eigenschaften von Trop-2 exprimierenden Zellen in vitro, während sich die invasive Fähigkeit der Transfektanten, in vitro durch eine Matrigelschicht zu wandern, nicht von den Vektorkontrollen unterschied. Die induzierte Expression von Trop-2 in der Mehrzahl von Metastasen des kolorektalen Karzinoms sowie in Tumoren von Lunge und Schilddrüse war eine neue Beobachtung. Diese Ergebnisse demonstrieren das Potential von Hochdurchsatzmethoden wie die hier verwendete Genchip-Technologie in Kombination mit geeigneten Zellmodellen. In der vorliegenden Arbeit führte sie zur Identifizierung von Trop-2 als einen Marker der Tumorprogression und Metastasierung von verschiedenen humanen Karzinomen.

Cancers of the gastrointestinal tract, including the liver, bile ducts, and pancreas, constitute the largest group of malignant tumors. Colorectal cancer is one of the most common neoplastic diseases in Western countries and one of the leading causes of cancer-related deaths. Inactivation of the adenomatous polyposis coli (APC) tumor-suppressor gene during early adenoma formation is thought to be the first genetic event in the process of colorectal carcinogenesis followed by mutations in oncogenes like K-Ras and tumor-suppressor genes like p53. Identification of the interaction of APC with the proto-oncogene beta-catenin has linked colorectal carcinogenesis to the Wnt-signal transduction pathway. The main function of APC is thought to be the regulation of free beta-catenin in concert with the glycogen synthase kinase 3beta (GSK-3beta) and Axin proteins. Loss of APC function, inactivation of Axin or activating beta-catenin mutations result in the cellular accumulation of beta-catenin. Upon translocation to the nucleus beta-catenin serves as an activator of T-cell factor (Tcf)-dependent transcription leading to an increased expression of several specific target genes including c-Myc, cyclin D1, MMP-7, and ITF-2. While APC mutations are almost exclusively found in colorectal cancers, deregulation of Wnt/beta-catenin/Tcf signaling is also common in other gastrointestinal and extra-gastrointestinal human cancers. In a fraction of hepatocellular carcinomas the Writ pathway is deregulated by inactivation of Axin or stabilizing mutations of beta-catenin. The majority of hepatoblastomas and a group of gastric cancers also carry beta-catenin mutations. Clearly, this pathway harbors great potential for future applications in cancer diagnostics, staging, and therapy. Copyright (C) 2002 S. Karger AG, Basel.