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| 2. |
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| 3. |
- de la Rosa, Andrés
(author)
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Design, expression, and analysis of antibody-based blood-brain barrier shuttles
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Antibody therapeutics, with their strong and highly selective target binding, are now used to treat various diseases. However, to enable their use to treat brain disorders, they must be delivered across the blood-brain barrier (BBB), as without active transport, only around 0.01% of intravenously injected doses reach the brain. Brain delivery can be done by BBB shuttles capable of binding receptors that naturally transport proteins, e.g., the Transferrin receptor (TfR). This thesis has studied strategies for designing TfR-binding shuttles and how to enhance the protein expression of antibody therapeutics. In Paper I, we shared our updated transient gene expression (TGE) protocol and developed a small-scale version to surmount the cost limitations of testing many conditions. Large variations of protein expression were observed for both protocols, prompting future studies investigating its cause(s). In paper II, we investigated if binding to the glycosaminoglycan heparan sulfate (HS) present at the BBB could improve brain delivery. Our results indicate that the BBB shuttle scFv8D3 is not dependent on the HS-binding sites identified, and adding new HS-binding sites did not enhance delivery. However, further studies are required due to HS's complexity and heterogeneity. Decreasing the TfR affinity of BBB shuttles has been shown to boost the delivery of therapeutic doses of high affinity anti-TfR antibodies, e.g., bivalent 8D3 antibodies. In Paper III, we applied the strategy to a monovalent single-chain fragment variable (scFv) of 8D3 (scFv8D3) based BBB shuttle. Our affinity mutants exhibited lowered TfR affinity, longer blood half-life, and higher brain concentration. Using our In-Cell BBB Trans assay, we concluded that the increased brain concentration is likely due to extended blood half-life. In paper IV, we fused the TfR ligand holo-transferrin to the TfR binding arms of the partly bivalent RmAb158-scFv8D3 antibody. Our results indicate that the TfR binding shifted from partly to fully bivalent, resulting in markedly decreased in vitro transcytosis. The potential transcytosis-promoting effect of the fused holoTf was absent and/or counteracted by the bivalent binding of the design. However, the strategy may still prove useful for monovalent TfR binders. In conclusion, monovalent and low-to-moderate affinity are likely beneficial binding properties for TfR-mediated brain delivery at therapeutic doses. However, whether it is possible to enhance brain delivery with HS-binding or holoTf-fusion requires further study.
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| 4. |
- de la Rosa, Andres, et al.
(author)
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Introducing or removing heparan sulfate binding sites does not alter brain uptake of the blood-brain barrier shuttle scFv8D3
- 2022
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Journal article (peer-reviewed)abstract
- The blood-brain barrier (BBB) greatly limits the delivery of protein-based drugs into the brain and is a major obstacle for the treatment of brain disorders. Targeting the transferrin receptor (TfR) is a strategy for transporting protein-based drugs into the brain, which can be utilized by using TfR-binding BBB transporters, such as the TfR-binding antibody 8D3. In this current study, we investigated if binding to heparan sulfate (HS) contributes to the brain uptake of a single chain fragment variable of 8D3 (scFv8D3). We designed and produced a scFv8D3 mutant, engineered with additional HS binding sites, HS(+)scFv8D3, to assess whether increased HS binding would improve brain uptake. Additionally, a mutant with a reduced number of HS binding sites, HS(-)scFv8D3, was also engineered to see if reducing the HS binding sites could also affect brain uptake. Heparin column chromatography showed that only the HS(+)scFv8D3 mutant bound HS in the experimental conditions. Ex vivo results showed that the brain uptake was unaffected by the introduction or removal of HS binding sites, which indicates that scFv8D3 is not dependent on the HS binding sites for brain uptake. Conversely, introducing HS binding sites to scFv8D3 decreased its renal excretion while removing them had the opposite effect.
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| 5. |
- Degerstedt, Oliver, et al.
(author)
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Quantitative imaging of doxorubicin diffusion and cellular uptake in biomimetic gels with human liver tumor cells
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Other publication (other academic/artistic)abstract
- Novel tumor-on-a-chip approaches are increasingly used to investigate tumor progression and potential treatment options. To improve the effect of any cancer treatment it is important to have an in-depth understanding of drug diffusion, penetration across the tumor extracellular matrix and cellular uptake. In this study, we have developed a miniaturized chip where drug diffusion and cellular uptake in different hydrogel environments can be quantified at high resolution using live imaging. Diffusion of doxorubicin was reduced in a biomimetic hydrogel mimicking tissue properties of cirrhotic liver and early stage hepatocellular carcinoma (362 ± 109 µm2/s) as compared to an agarose gel (571 ± 145 µm2/s, p = 0.0085). The diffusion was further lowered to 164 ± 33 µm2/s (p = 0.0023) by preparing the biomimetic gel in cell media instead of phosphate buffered saline. The addition of liver tumor cells (Huh7 or HepG2) to the gel, at two different densities, did not significantly influence drug diffusion. Clinically relevant and quantifiable doxorubicin concentration gradients (1-20 µM) were established in the chip within one hour. Intracellular increases in doxorubicin fluorescence correlated with decreasing fluorescence of the DNA-binding stain Hoechst 33342, and based on the quantified intracellular uptake of doxorubicin an apparent cell permeability (9.00 ± 0.74 x 10-4 µm/s for HepG2) was determined.
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| 6. |
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| 7. |
- Echeverri Correa, Estefania, et al.
(author)
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Fe and C additions decrease the dissolution rate of silicon nitride coatings and are compatible with microglial viability in 3D collagen hydrogels
- 2023
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In: Biomaterials Science. - : Royal Society of Medicine Press. - 2047-4830 .- 2047-4849. ; 11:9, s. 3144-3158
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Journal article (peer-reviewed)abstract
- Silicon nitride (SiN) coatings may reduce unwanted release of metal ions from metallic implants. However, as SiN slowly dissolves in aqueous solutions, additives that reduce this dissolution rate would likely increase the lifetime and functionality of implants. Adding iron (Fe) and carbon (C) permits tuning of the SiN coatings’ mechanical properties, but their effect on SiN dissolution rates, and their capacity to reduce metal ion release from metallic implant substrates, have yet to be investigated. Such coatings have recently been proposed for use in spinal implants; therefore, it is relevant to assess their impact on the viability of cells expected at the implant site, such as microglia, the resident macrophages of the central nervous system (CNS). To study the effects of Fe and C on the dissolution rate of SiN coatings, compositional gradients of Si, Fe and C in combination with N were generated by physical vapor deposition onto CoCrMo discs. Differences in composition did not affect the surface roughness or the release of Si, Fe or Co ions (the latter from the CoCrMo substrate). Adding Fe and C reduced ion release compared to a SiN reference coating, which was attributed to altered reactivity due to an increase in the fraction of stabilizing Si–C or Fe–C bonds. Extracts from the SiN coatings containing Fe and C were compatible with microglial viability in 2D cultures and 3D collagen hydrogels, to a similar degree as CoCrMo and SiN coated CoCrMo reference extracts. As Fe and C reduced the dissolution rate of SiN-coatings and did not compromise microglial viability, the capacity of these additives to extend the lifetime and functionality of SiN-coated metallic implants warrants further investigation.
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| 8. |
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| 9. |
- Echeverri Correa, Estefania, et al.
(author)
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In vitro 3D model for monitoring glial cell responses to particles and ions released from spinal implants
- 2023
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Conference paper (peer-reviewed)abstract
- Spinal implants have been used for decades to treat different spinal conditions. However, certain implant-related complications have been attributed to the release of particles and ions due to corrosion and wear triggering local immune responses including the release of pro-inflammatory cytokines, leading to local inflammation. The impact of these particles and ions on cells from the central nervous system (CNS) remains largely unknown, with few studies examining the effects on glial cells1. Indeed, the particles may migrate to adjacent nervous tissues and increasing our knowledge of the glial cell response is essential since they play a crucial role in maintaining tissue homeostasis and protecting the CNS. Most prior studies have used traditional 2D culture models; however, these lack the 3D spatial arrangement of cells found in tissues where they form important interactions with the extracellular matrix. The aim of this study was to employ an open source bioprinter2 to extrude hydrogels containing glial cells into which experimental implant debris can be introduced, enabling monitoring of cell viability and inflammatory responses by fluorescence microscopy. We have previously established that mono-cultures of microglia and astrocytes can be 3D cultured in collagen hydrogels, and their viability monitored using the caspase-3/7 apoptosis reporter and propidium iodide labelling for cell death. Applying a bioprinting strategy to produce these glial-laden constructs increases the reproducibility of these models, and allows the study of a wide range of types and concentrations of particles, resulting in a valuable tool to increase the knowledge about the biological response generated by particles from spinal implants.
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| 10. |
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| 11. |
- Engberg, Adam, et al.
(author)
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An open source extrusion bioprinter based on the E3D motion system and tool changer to enable FRESH and multimaterial bioprinting
- 2021
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Journal article (peer-reviewed)abstract
- Bioprinting is increasingly used to create complex tissue constructs for an array of research applications, and there are also increasing efforts to print tissues for transplantation. Bioprinting may also prove valuable in the context of drug screening for personalized medicine for treatment of diseases such as cancer. However, the rapidly expanding bioprinting research field is currently limited by access to bioprinters. To increase the availability of bioprinting technologies we present here an open source extrusion bioprinter based on the E3D motion system and tool changer to enable high-resolution multimaterial bioprinting. As proof of concept, the bioprinter is used to create collagen constructs using freeform reversible embedding of suspended hydrogels (FRESH) methodology, as well as multimaterial constructs composed of distinct sections of laminin and collagen. Data is presented demonstrating that the bioprinted constructs support growth of cells either seeded onto printed constructs or included in the bioink prior to bioprinting. This open source bioprinter is easily adapted for different bioprinting applications, and additional tools can be incorporated to increase the capabilities of the system.
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| 12. |
- Fatsis-Kavalopoulos, Nikos, et al.
(author)
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Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
- 2019
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In: Lab on a Chip. - : ROYAL SOC CHEMISTRY. - 1473-0197 .- 1473-0189. ; 19:6, s. 1071-1081
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Journal article (peer-reviewed)abstract
- The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.
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| 13. |
- Gaulton, Kyle J, et al.
(author)
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Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.
- 2015
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In: Nature Genetics. - : Springer Science and Business Media LLC. - 1546-1718 .- 1061-4036. ; 47:12, s. 1415-1415
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Journal article (peer-reviewed)abstract
- We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.
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| 14. |
- Gustavsson, Tobias, et al.
(author)
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SPECT imaging of distribution and retention of a brain-penetrating bispecific amyloid-beta antibody in a mouse model of Alzheimer's disease
- 2020
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In: Translational Neurodegeneration. - : BMC. - 2047-9158. ; 9:1
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Journal article (peer-reviewed)abstract
- Background Alzheimer's disease (AD) immunotherapy with antibodies targeting amyloid-beta (A beta) has been extensively explored in clinical trials. The aim of this study was to study the long-term brain distribution of two radiolabeled monoclonal A beta antibody variants - RmAb158, the recombinant murine version of BAN2401, which has recently demonstrated amyloid removal and reduced cognitive decline in AD patients, and the bispecific RmAb158-scFv8D3, which has been engineered for enhanced brain uptake via transferrin receptor-mediated transcytosis. Methods A single intravenous injection of iodine-125 (I-125)-labeled RmAb158-scFv8D3 or RmAb158 was administered to AD transgenic mice (tg-ArcSwe). In vivo single-photon emission computed tomography was used to investigate brain retention and intrabrain distribution of the antibodies over a period of 4 weeks. Activity in blood and brain tissue was measured ex vivo and autoradiography was performed in combination with A beta and CD31 immunostaining to investigate the intrabrain distribution of the antibodies and their interactions with A beta. Results Despite faster blood clearance, [I-125]RmAb158-scFv8D3 displayed higher brain exposure than [I-125]RmAb158 throughout the study. The brain distribution of [I-125]RmAb158-scFv8D3 was more uniform and coincided with parenchymal A beta pathology, while [I-125]RmAb158 displayed a more scattered distribution pattern and accumulated in central parts of the brain at later times. Ex vivo autoradiography indicated greater vascular escape and parenchymal A beta interactions for [I-125]RmAb158-scFv8D3, whereas [I-125]RmAb158 displayed retention and A beta interactions in lateral ventricles. Conclusions The high brain uptake and uniform intrabrain distribution of RmAb158-scFv8D3 highlight the benefits of receptor-mediated transcytosis for antibody-based brain imaging. Moreover, it suggests that the alternative transport route of the bispecific antibody contributes to improved efficacy of brain-directed immunotherapy.
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| 15. |
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| 16. |
- Heldin, Johan, et al.
(author)
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FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation
- 2017
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In: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 40, s. 125-132
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Journal article (peer-reviewed)abstract
- The complete repertoire of endothelial functions elicited by FGD5, a guanine nucleotide exchange factor activating the Rho GTPase Cdc42, has yet to be elucidated. Here we explore FGD5's importance during vascular endothelial growth factor A (VEGFA) signaling via VEGF receptor 2 (VEGFR2) in human endothelial cells. In microvascular endothelial cells, FGD5 is located at the inner surface of the cell membrane as well as at the outer surface of EEAl-positive endosomes carrying VEGFR2. The latter finding prompted us to explore if FGD5 regulates VEGFR2 dynamics. We found that depletion of FGD5 in microvascular cells inhibited their migration towards a stable VEGFA gradient. Furthermore, depletion of FGD5 resulted in accelerated VEGFR2 degradation, which was reverted by lactacystin-mediated proteasomal inhibition. Our results thus suggest a mechanism whereby FGD5 sustains VEGFA signaling and endothelial cell chemotaxis via inhibition of proteasome-dependent VEGFR2 degradation.
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| 17. |
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| 18. |
- Hernández Vera, Rodrigo, et al.
(author)
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Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures
- 2019
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
-
Journal article (peer-reviewed)abstract
- Microfluidic systems are very useful for in vitro studies of interactions between blood cells and vascular endothelial cells under flow, and several commercial solutions exist. However, the availability of customizable, user-designed devices is largely restricted to researchers with expertise in photolithography and access to clean room facilities. Here we describe a strategy for producing tailor-made modular microfluidic systems, cast in PDMS from 3D-printed molds, to facilitate studies of leukocyte adherence to endothelial cells. A dual-chamber barrier module was optimized for culturing two endothelial cell populations, separated by a 250 μm wide dividing wall, on a glass slide. In proof-of-principle experiments one endothelial population was activated by TNFα, while the other served as an internal control. The barrier module was thereafter replaced with a microfluidic flow module, enclosing both endothelial populations in a common channel. A suspension of fluorescently-labeled leukocytes was then perfused through the flow module and leukocyte interactions with control and tnfα-treated endothelial populations were monitored in the same field of view. Time-lapse microscopy analysis confirmed the preferential attachment of leukocytes to the TNFα-activated endothelial cells. We conclude that the functionality of these modular microfluidic systems makes it possible to seed and differentially activate adherent cell types, and conduct controlled side-by-side analysis of their capacity to interact with cells in suspension under flow. Furthermore, we outline a number of practical considerations and solutions associated with connecting and switching between the microfluidic modules, and the advantages of simultaneously and symmetrically analyzing control and experimental conditions in such a microfluidic system.
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| 19. |
- Kasza, Z., et al.
(author)
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MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)
- 2013
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:36, s. 25956-25963
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Journal article (peer-reviewed)abstract
- Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.
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| 20. |
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| 21. |
- Kreuger, Johan, et al.
(author)
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Failure to Genotype : A Cautionary Note on an Elusive loxP Sequence
- 2016
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:10
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Journal article (peer-reviewed)abstract
- Here we report on a technical difficulty we encountered while optimizing genotyping strategies to identify mice derived from Exoc3l2(tm1a( KOMP)Wtsi) embryonic stem cells obtained from the Knockout Mouse Project Repository. The Exoc3l2(tm1a(KOMP)Wtsi) construct encodes a "knockout-first" design with loxP sites that confer conditional potential (KO1st). We designed primers that targeted wild-type sequences flanking the most downstream element of the construct, an 80 base pair synthetic loxP region, which BLAST alignment analysis reveals is an element common to over 10,000 conditional gene-targeting mouse models. As PCR products amplified from KO1st and wild-type templates would have different lengths (and different mobility in an agarose gel) this strategy was designed to determine the zygosity of individual mice from a single PCR. In parallel we performed PCR with a primer specifically targeting the synthetic loxP sequence. Unexpectedly, while the latter strategy detected the synthetic loxP region and correctly genotyped KO1st chimeric mice, the same individuals were genotyped as wild-type when using the primers that flanked the synthetic loxP region. We discuss the possibility that secondary DNA structures, formed due to the palindromic nature of the synthetic loxP region, may have caused the KO1st template to elude the PCR when using primers that flanked this region. This brief report aims to raise awareness regarding this potential source of false-negative genotype results, particularly for those who are devising genotyping strategies for similarly engineered animal models.
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| 22. |
- Noborn, Fredrik, et al.
(author)
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Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein
- 2011
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:14, s. 5584-5589
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Journal article (peer-reviewed)abstract
- Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.
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| 23. |
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| 24. |
- O'Callaghan, Paul, et al.
(author)
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Apolipoprotein-E increases cell-associated amyloid-β through a heparan sulfate-dependent pathway
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Other publication (other academic/artistic)abstract
- The increased risk of Alzheimer’s disease (AD) associated with specific apolipoprotein E (ApoE) isoforms appears to relate to altered amyloid-β (Aβ) homeostasis. The efficiency of Aβ clearance from the brain is reduced in the presence of the AD-associated ApoE4 isoform, which may explain the accumulation of Aβ deposits in the parenchyma and vasculature. The low density lipoprotein receptor-related protein 1 (LRP1) and heparan sulfate proteoglycans (HSPGs) are involved in Aβ uptake, with LRP1 further implicated in Aβ transcytosis across the blood brain barrier. However, both are also established ApoE receptors and function co-operatively to mediate cell interactions with lipoproteins and Aβ. Here we determined that HS, ApoE and LRP1 co-occur in Aβ40-positive microvessels of AD brain, establishing the relevance of studying interactions between these molecules. Using Chinese hamster ovary (CHO) cells deficient in HS or LRP1 we found that ApoE increases the levels of cell-associated Aβ in primarily a HSPG-dependent manner. Furthermore, in this model we found that ApoE is alternatively processed in the absence of cell surface HS, supporting a role for HSPGs in ApoE metabolism. The findings presented here raise the possibility that ApoE can increase Aβ associations with HSPGs and LRP1 in the vasculature. This may facilitate clearance, but if unbalanced could contribute to Aβ accumulation and the pathogenesis of AD.
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| 25. |
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| 26. |
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| 27. |
- O'Callaghan, Paul, et al.
(author)
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Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells
- 2008
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In: Brain Pathology. - : Wiley. - 1015-6305 .- 1750-3639. ; 18:4, s. 548-561
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Journal article (peer-reviewed)abstract
- Amyloid beta-peptide (Abeta) plaques, one of the major neuropathological lesions in Alzheimer's disease (AD), can be broadly subdivided into two morphological categories: neuritic and diffuse. Heparan sulfate (HS) and HS proteoglycans (HSPGs) are codeposits of multiple amyloidoses, including AD. Although HS has been considered a limiting factor in the initiation of amyloid deposition, the pathological implications of HS in Abeta deposits of AD remain unclear. In this study, immunohistochemistry combined with fluorescence and confocal microscopy was employed to gain deeper insight into the accumulation of HS with Abeta plaques in sporadic and familial AD. Here we demonstrate that HS preferentially accumulated around the Abeta40 dense cores of neuritic plaques, but was largely absent from diffuse Abeta42 plaques, suggesting that Abeta42 deposition may occur independently of HS. A codeposition pattern of HS with Abeta deposits in Tg2576 mice was also examined. We identified the membrane-bound HSPGs, glypican-1 (GPC1) and syndecan-3 (SDC3), in glial cells associated with Abeta deposits, proximal to sites of HS accumulation. In mouse primary glial cultures, we observed increased levels of GPC1 and SDC3 following Abeta stimulation. These results suggest that HS codeposits with Abeta40 in neuritic plaques and is mainly derived from glial cells.
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| 28. |
- O'Callaghan, Paul
(author)
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Heparan Sulfate in the Amyloidosis and Inflammation of Alzheimer’s Disease
- 2011
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Doctoral thesis (other academic/artistic)abstract
- Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD. In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation. Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier.The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.
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| 29. |
- O'Callaghan, Paul, et al.
(author)
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Heparan Sulfate Proteoglycans as Relays of Neuroinflammation
- 2018
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In: Journal of Histochemistry and Cytochemistry. - : SAGE PUBLICATIONS LTD. - 0022-1554 .- 1551-5044. ; 66:4, s. 305-319
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Research review (peer-reviewed)abstract
- Heparan sulfate proteoglycans (HSPGs) are implicated as inflammatory mediators in a variety of settings, including chemokine activation, which is required to recruit circulating leukocytes to infection sites. Heparan sulfate (HS) polysaccharide chains are highly interactive and serve co-receptor roles in multiple ligand:receptor interactions. HS may also serve as a storage depot, sequestering ligands such as cytokines and restricting their access to binding partners. Heparanase, through its ability to fragment HS chains, is a key regulator of HS function and has featured prominently in studies of HS's involvement in inflammatory processes. This review focuses on recent discoveries regarding the role of HSPGs, HS, and heparanase during inflammation, with particular focus on the brain. HS chains emerge as critical go-betweens in multiple aspects of the inflammatory responserelaying signals between receptors and cells. The molecular interactions proposed to occur between HSPGs and the pathogen receptor toll-like receptor 4 (TLR4) are discussed, and we summarize some of the contrasting roles that HS and heparanase have been assigned in diseases associated with chronic inflammatory states, including Alzheimer's disease (AD). We conclude by briefly discussing how current knowledge could potentially be applied to augment HS-mediated events during sustained neuroinflammation, which contributes to neurodegeneration in AD.
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| 30. |
- O'Callaghan, Paul, et al.
(author)
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Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response
- 2015
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:24, s. 14904-14914
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Journal article (peer-reviewed)abstract
- Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1β (IL1β) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1β. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism.
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| 31. |
- O'Callaghan, Paul, et al.
(author)
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Microglial heparan sulfate proteoglycans mediate pro-inflammatory signaling
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Other publication (other academic/artistic)abstract
- Microglia are the central nervous system’s (CNS) first line of defense against pathogenic insults and acute inflammatory responses are necessary for the resolution of infection. However, unregulated and/or chronic activation of microglia is associated with neurodegeneration. Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation, but the possibility that HSPGs are integral to pro-inflammatory signaling mechanisms has not been fully explored. To analyze the relevance of microglial HSPGs in the pro-inflammatory response we isolated primary microglia from mice overexpressing human heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with the pro-inflammatory endotoxin lipopolysaccharide (LPS). The LPS-induced upregulation of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was inhibited in Hpa-tg microglia, as was upregulation of the LPS-receptor CD14. Analysis of HSPG structures revealed that Hpa-tg microglia produced truncated HS chains. Importantly, co-treatment of microglia with heparin attenuated LPS-induced cytokine upregulation. Together these findings implicate microglial HSPGs as key facilitators of the pro-inflammatory response. Astrocytes constitute a critical support network in the CNS, but are also implicated in inflammation. LPS induced comparable levels of TNF-α in Hpa-tg and Ctrl astrocytes, indicating that the mechanism of HSPG-dependent inflammation is specific to microglia. We conclude that microglial HSPGs are required for pro-inflammatory signaling events and that heparanase, through its HS-degrading activity, can regulate this mechanism.
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| 32. |
- O'Callaghan, Paul, et al.
(author)
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Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec
- 2018
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In: Plos One. - San Francisco, California, US : Public Library of Science (PLoS). - 1932-6203. ; 13:8
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Journal article (peer-reviewed)abstract
- The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst.
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| 33. |
- O'Callaghan, Paul, et al.
(author)
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Piezo1 activation attenuates thrombin-induced blebbing in breast cancer cells
- 2022
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 135:7
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Journal article (peer-reviewed)abstract
- Cancer cells exploit a variety of migration modes to leave primary tumors and establish metastases, including amoeboid cell migration, which is typically reliant on bleb formation. Here we demonstrate that thrombin induces dynamic blabbing in the MDA-MB-231 breast cancer cell line and confirm that protease-activated receptor 1 (PAR1) activation is sufficient to induce this effect. Cell confinement has been implicated as a driving force in bleb-based migration. Unexpectedly, we found that gentle contact compression, exerted using a custom built 'cell press' to mechanically stimulate cells, reduced thrombin-induced blebbing. Thrombin-induced blebbing was similarly attenuated using the small molecule Yoda1, an agonist of the mechanosensitive Ca2+ channel Piezo1, and this attenuation was impaired in Piezo1-depleted cells. Additionally, Piezo1 activation suppressed thrombin-induced phosphorylation of ezrin, radixin and moesin (ERM) proteins, which are implicated in the blabbing process. Our results provide mechanistic insights into Piezo1 activation as a suppressor of dynamic blebbing, specifically that which is induced by thrombin.
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| 34. |
- Philipson, Ola, et al.
(author)
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Animal models of amyloid-β-related pathologies in Alzheimer’s disease
- 2010
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In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:6, s. 1389-1409
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Research review (peer-reviewed)abstract
- In the early 1990s, breakthrough discoveries on the genetics of Alzheimer's disease led to the identification of missense mutations in the amyloid-beta precursor protein gene. Research findings quickly followed, giving insights into molecular pathogenesis and possibilities for the development of new types of animal models. The complete toolbox of transgenic techniques, including pronuclear oocyte injection and homologous recombination, has been applied in the Alzheimer's disease field, to produce overexpressors, knockouts, knockins and regulatable transgenics. Transgenic models have dramatically advanced our understanding of pathogenic mechanisms and allowed therapeutic approaches to be tested. Following a brief introduction to Alzheimer's disease, various nontransgenic and transgenic animal models are described in terms of their values and limitations with respect to pathogenic, therapeutic and functional understandings of the human disease.
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| 35. |
- Rostami, Jinar, et al.
(author)
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Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-β aggregates
- 2021
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In: Journal of Neuroinflammation. - : Springer Science and Business Media LLC. - 1742-2094. ; 18:1
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Journal article (peer-reviewed)abstract
- BackgroundAlzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aβ) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aβ/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear.MethodsThe aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aβ pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aβ and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized “close-culture” chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space.ResultsOur data show that intracellular deposits of αSYN and Aβ are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the “close-culture” chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte.ConclusionsTaken together, our data demonstrate the importance of astrocyte and microglia interactions in Aβ/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.
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| 36. |
- Sanchez, Gonzalo, et al.
(author)
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The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signaling
- 2023
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In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 222:1
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Journal article (peer-reviewed)abstract
- The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting β-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet β-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.
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| 37. |
- Sandwall, Elina, et al.
(author)
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Heparan sulfate mediates amyloid-beta internalization and cytotoxicity
- 2010
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In: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 20:5, s. 533-541
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Journal article (peer-reviewed)abstract
- Heparan sulfate (HS) has been found associated with amyloid deposits, including the toxic amyloid-beta (Abeta) peptide aggregates in cerebral vasculature and neuronal tissues in patients with Alzheimer's disease. However, the pathophysiological significance of the HS-Abeta interaction has remained unclear. In the present study, we applied cell models to gain insight into the roles of HS in relation to Abeta toxicity. Wild-type Chinese hamster ovary (CHO-WT) cells showed loss of viability following exposure to Abeta40, whereas the HS-deficient cell line, pgsD-677, was essentially resistant. Immunocytochemical analysis showed Abeta internalization by CHO-WT, but not pgsD-677 cells. Abeta40 toxicity was also attenuated in human embryonic kidney cells overexpressing heparanase. Finally, addition of heparin to human umbilical vein endothelial cells prevented internalization of added Abeta40 and protected against Abeta toxicity. Taken together, these findings suggest that cell-surface HS mediates Abeta internalization and toxicity.
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| 38. |
- Tobias, Gustavsson, 1985-
(author)
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Brain distribution of a bispecific antibody targeting Aβ
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Alzheimer’s disease (AD) is characterised by aberrant protein aggregation in the brain with subsequent synaptic loss, neuroinflammation, and brain atrophy that ultimately clinically manifests as cognitive impairment. Histopathological findings in AD are extracellular plaques of the protein amyloid-beta (Aβ), Aβ in blood vessels (CAA), and intracellular neurofibrillary tangles (NFT) of hyperphosphorylated tau. The FDA recently approved the antibody aducanumab for AD treatment, and several antibodies are now in clinical phase 3 trials, demonstrating that Aβ-directed immunotherapy is a viable treatment option in AD. In this thesis we evaluated the therapeutic Aβ antibodies 3D6 and RmAb158 in comparison with the bispecific RmAb158-scFv8D3, which penetrates the blood-brain barrier (BBB) by transferrin receptor mediated transcytosis. Emphasis lies in antibody brain uptake and intra brain distribution, in their use as potential treatment options in AD and how such treatment affects BBB integrity. Vascular disturbances are common side effects of anti-Aβ immunotherapy. However, we demonstrated that the BBB permeability of large molecules is unchanged following acute 3D6 treatment in an Aβ mouse model (paper I). Next, brain uptake and distribution of radioiodinated RmAb158 and its bispecific variant RmAb158-scFv8D3 were investigated with SPECT in an Aβ mouse model. Due to its active transport across BBB, RmAb158-scFv8D3 had a higher brain uptake than RmAb158, resulting in greater total brain exposure, and higher concentration at Aβ plaques (paper II). In paper III, we labelled RmAb158-scFv8D3 with the radiometal indium-111 (111In), using chelators CHX-A”-DTPA or DOTA, and SPECT was used to investigate brain retention and biodistribution. The 111In-labelled bispecific antibody entered the brain, and although brain retention was higher in Aβ mice, the wild type (wt) background was high. SPECT revealed high bone uptake of all tracers, and subsequent ex vivo measurement pinpointed retention to the bone marrow. With the knowledge gained from paper II, we addressed whether RmAb158-scFv8D3 would improve treatment efficacy in different treatment regimes. We also assessed the immunogenicity of different antibody constructs upon chronic administration (paper IV). As all tested bispecific antibody constructs elicited a humoral response, immune cell depletion was necessary before repeated antibody treatment. Overall, long-term treatment of RmAb158-scFv8D3 did lower total brain Aβ but compared with RmAb158, it did not improve treatment efficacy.In conclusion, acute anti-Aβ immunotherapy did not negatively affect BBB integrity, and bispecific antibodies displayed improved brain distribution and long-term accumulation at parenchymal Aβ. However, this did not translate into an added treatment effect in a chronic therapeutic setting.
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| 39. |
- Zhang, Xiao, et al.
(author)
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Heparanase overexpression impairs inflammatory response and macrophage-mediated clearance of amyloid-beta in murine brain
- 2012
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In: Acta Neuropathologica. - : Springer Science and Business Media LLC. - 0001-6322 .- 1432-0533. ; 124:4, s. 465-478
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Journal article (peer-reviewed)abstract
- Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimer's disease, as well as after traumatic injury and pathogen infection. Resident immune cells, microglia and astrocytes, are activated and joined by blood-borne monocytes that traverse the blood-brain barrier and convert into activated macrophages. The activated cells express various cytokines, chemokines and proteolytic enzymes. To study the role of heparan sulfate proteoglycans in neuroinflammation, we employed a transgenic mouse overexpressing heparanase, an endoglucuronidase that specifically degrades heparan sulfate side chains. Neuroinflammation was induced by systemic challenge with lipopolysaccharide, or by localized cerebral microinjection of aggregated amyloid-beta peptide, implicated in Alzheimer's disease. Lipopolysaccharide-treated control mice showed massive activation of resident microglia as well as recruitment of monocyte-derived macrophages into the brain parenchyma. Microinjection of aggregated amyloid-beta elicited a similar inflammatory response, albeit restricted to the injection site, which led to dispersion and clearance of the amyloid. In the heparanase-overexpressing mice, all aspects of immune cell recruitment and activation were significantly attenuated in both inflammation models, as was amyloid dispersion. Accordingly, an in vitro blood-brain barrier model constructed from heparanase-overexpressing cerebral vascular cells showed impaired transmigration of monocytes compared to a corresponding assembly of control cells. Our data indicate that intact heparan sulfate chains are required at multiple sites to mediate neuroinflammatory responses, and further point to heparanase as a modulator of this process, with potential implications for Alzheimer's disease.
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| 40. |
- Zhang, Xiao, et al.
(author)
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Heparanase overexpression impedes perivascular clearance of amyloid-beta from murine brain : relevance to Alzheimer's disease
- 2021
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In: Acta neuropathologica communications. - : BioMed Central (BMC). - 2051-5960. ; 9
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Journal article (peer-reviewed)abstract
- Defective amyloid-beta (A beta) clearance from the brain is a major contributing factor to the pathophysiology of Alzheimer's disease (AD). A beta clearance is mediated by macrophages, enzymatic degradation, perivascular drainage along the vascular basement membrane (VBM) and transcytosis across the blood-brain barrier (BBB). AD pathology is typically associated with cerebral amyloid angiopathy due to perivascular accumulation of A beta. Heparan sulfate (HS) is an important component of the VBM, thought to fulfill multiple roles in AD pathology. We previously showed that macrophage-mediated clearance of intracortically injected A beta was impaired in the brains of transgenic mice overexpressing heparanase (Hpa-tg). This study revealed that perivascular drainage was impeded in the Hpa-tg brain, evidenced by perivascular accumulation of the injected A beta in the thalamus of Hpa-tg mice. Furthermore, endogenous A beta accumulated at the perivasculature of Hpa-tg thalamus, but not in control thalamus. This perivascular clearance defect was confirmed following intracortical injection of dextran that was largely retained in the perivasculature of Hpa-tg brains, compared to control brains. Hpa-tg brains presented with thicker VBMs and swollen perivascular astrocyte endfeet, as well as elevated expression of the BBB-associated water-pump protein aquaporin 4 (AQP4). Elevated levels of both heparanase and AQP4 were also detected in human AD brain. These findings indicate that elevated heparanase levels alter the organization and composition of the BBB, likely through increased fragmentation of BBB-associated HS, resulting in defective perivascular drainage. This defect contributes to perivascular accumulation of A beta in the Hpa-tg brain, highlighting a potential role for heparanase in the pathogenesis of AD.
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