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Publications (10 of 10) Show all publications
Hinojosa, M. G., Johansson, Y., Cediel-Ulloa, A., Ivanova, E. V., Gabring, N., Gliga, A. & Forsby, A. (2023). Evaluation of mRNA markers in differentiating human SH-SY5Y cells for estimation of developmental neurotoxicity. Neurotoxicology, 97, 65-77
Open this publication in new window or tab >>Evaluation of mRNA markers in differentiating human SH-SY5Y cells for estimation of developmental neurotoxicity
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2023 (English)In: Neurotoxicology, ISSN 0161-813X, E-ISSN 1872-9711, Vol. 97, p. 65-77Article in journal (Refereed) Published
Abstract [en]

Current guidelines for developmental neurotoxicity (DNT) evaluation are based on animal models. These have limitations so more relevant, efficient and robust approaches for DNT assessment are needed. We have used the human SH-SY5Y neuroblastoma cell model to evaluate a panel of 93 mRNA markers that are frequent in Neuronal diseases and functional annotations and also differentially expressed during retinoic acid-induced differentiation in the cell model. Rotenone, valproic acid (VPA), acrylamide (ACR) and methylmercury chloride (MeHg) were used as DNT positive compounds. Tolbutamide, D-mannitol and clofibrate were used as DNT negative compounds. To determine concentrations for exposure for gene expression analysis, we developed a pipeline for neurite outgrowth assessment by live-cell imaging. In addition, cell viability was measured by the resazurin assay. Gene expression was analyzed by RT-qPCR after 6 days of exposure during differentiation to concentrations of the DNT positive compounds that affected neurite outgrowth, but with no or minimal effect on cell viability. Methylmercury affected cell viability at lower concentrations than neurite outgrowth, hence the cells were exposed with the highest non-cytotoxic concentration. Rotenone (7.3 nM) induced 32 differentially expressed genes (DEGs), ACR (70 µM) 8 DEGs, and VPA (75 µM) 16 DEGs. No individual genes were significantly dysregulated by all 3 DNT positive compounds (p < 0.05), but 9 genes were differentially expressed by 2 of them. Methylmercury (0.8 nM) was used to validate the 9 DEGs. The expression of SEMA5A (encoding semaphorin 5A) and CHRNA7 (encoding nicotinic acetylcholine receptor subunit α7) was downregulated by all 4 DNT positive compounds. None of the DNT negative compounds dysregulated any of the 9 DEGs in common for the DNT positive compounds. We suggest that SEMA5A or CHRNA7 should be further evaluated as biomarkers for DNT studies in vitro since they also are involved in neurodevelopmental adverse outcomes in humans.

Keywords
Developmental neurotoxicity, in vitro, mRNA markers, Live-cell imaging, Neurite outgrowth
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-229648 (URN)10.1016/j.neuro.2023.05.011 (DOI)001010188500001 ()37210002 (PubMedID)2-s2.0-85160084104 (Scopus ID)
Available from: 2024-05-27 Created: 2024-05-27 Last updated: 2024-05-27Bibliographically approved
Koistinen, N. A., Edlund, A. K., Menon, P. K., Ivanova, E. V., Bacanu, S. & Iverfeldt, K. (2017). Nuclear localization of amyloid-beta precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis. PLOS ONE, 12(3), Article ID e0173888.
Open this publication in new window or tab >>Nuclear localization of amyloid-beta precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis
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2017 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 12, no 3, article id e0173888Article in journal (Refereed) Published
Abstract [en]

Fe65 is an adaptor protein involved in both processing and signaling of the Alzheimer-associated amyloid-beta precursor protein, APP. Here, the subcellular localization was further investigated using TAP-tagged Fe65 constructs expressed in human neuroblastoma cells. Our results indicate that PTB2 rather than theWWdomain is important for the nuclear localization of Fe65. Electrophoretic mobility shift of Fe65 caused by phosphorylation was not detected in the nuclear fraction, suggesting that phosphorylation could restrict nuclear localization of Fe65. Furthermore, both ADAM10 and gamma-secretase inhibitors decreased nuclear Fe65 in a similar way indicating an important role also of alpha-secretase in regulating nuclear translocation.

National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-143465 (URN)10.1371/journal.pone.0173888 (DOI)000399089000040 ()28323844 (PubMedID)
Available from: 2017-06-02 Created: 2017-06-02 Last updated: 2022-03-23Bibliographically approved
Ivanova, E. V., Figueroa, R. A., Gatsinzi, T., Hallberg, E. & Iverfeldt, K. (2016). Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution. Sensors, 16(5), Article ID 703.
Open this publication in new window or tab >>Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution
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2016 (English)In: Sensors, E-ISSN 1424-8220, Vol. 16, no 5, article id 703Article in journal (Refereed) Published
Abstract [en]

FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling.

Keywords
apoptosis, caspase, FRET sensor, live cell imaging, neurodegeneration, signal transduction
National Category
Biological Sciences Biomedical Laboratory Science/Technology Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-132518 (URN)10.3390/s16050703 (DOI)000378510400074 ()
Available from: 2016-08-15 Created: 2016-08-15 Last updated: 2022-03-23Bibliographically approved
Ivanova, E. V. (2015). Caspase activation in human neuroblastoma cells: mechanisms and spatiotemporal aspects. (Licentiate dissertation). Stockholm: Department of Neurochemistry
Open this publication in new window or tab >>Caspase activation in human neuroblastoma cells: mechanisms and spatiotemporal aspects
2015 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis is one of the modes of programmed cell death, in which several members of the caspase family of proteases play the central role. However, activation of apoptotic caspases does not necessarily lead to cell death. Instead, these caspases may mediate, for instance, differentiation or synaptic plasticity, if their activity is restricted in space and time. Such localized caspase activation has been also implicated in the initial stages of neurodegeneration. In order to assess this kind of events at a subcellular level, our research group has previously constructed tau-anchored FRET-based caspase sensors (tAFSs). Here, we demonstrate that localization of tAFSs to the cytoskeleton results in enrichment of the sensors in neuritic processes and enables increased spatiotemporal resolution for live cell imaging of caspase activation, as compared to soluble FRET sensors. This feature is particularly beneficial for investigation of neurodegeneration-related processes.

tAFSs were further employed for investigation of caspase activation in neuroblastoma, an extracranial solid pediatric tumor. Tumor necrosis factor-related apoptosis inducing factor (TRAIL) is a promising candidate for cancer treatment due to its ability to selectively trigger apoptosis malignant cells. However, many cancer cells, including neuroblastoma, acquire resistance to TRAIL. Here, we show that in S-type neuroblastoma cell lines, TRAIL resistance is dependent on incomplete activation of apoptotic caspase-3. Sensitization to TRAIL was achieved with protein kinase C (PKC)-inhibiting compounds, suggesting a role for this kinase in blocking the apoptotic response to TRAIL. This effect of PKC could possibly involve stabilization of XIAP, an endogenous caspase inhibitor, as PKC inhibition, in combination with TRAIL treatment, led to downregulation of XIAP.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, 2015
Keywords
apoptosis, caspase, local caspase activation, neurodegeneration, tau-anchored FRET sensors, tAFS, cancer, neuroblastoma, TNF-related apoptosis inducing ligand, TRAIL, protein kinase C, PKC, X-linked inhibitor of apoptosis protein, XIAP
National Category
Chemical Sciences Biochemistry and Molecular Biology Cell Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-122651 (URN)978-91-7649-303-8 (ISBN)
Presentation
2015-11-27, Heilbronnsalen, C458, Svante Arrhenius väg 16B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2015-11-12 Created: 2015-11-05 Last updated: 2022-02-23Bibliographically approved
Tracy, L., Bergqvist, F., Ivanova, E., Jacobsen, K. & Iverfeldt, K. (2013). Exposure to the Saturated Free Fatty Acid Palmitate AltersBV-2 Microglia Inflammatory Response. Journal of Molecular Neuroscience, 51(3), 805-812
Open this publication in new window or tab >>Exposure to the Saturated Free Fatty Acid Palmitate AltersBV-2 Microglia Inflammatory Response
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2013 (English)In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 51, no 3, p. 805-812Article in journal (Refereed) Published
Abstract [en]

Elevated levels of free fatty acids (FFAs) in plasma and increased incidence of chronic systemic inflammation are associated with obesity. In the brain, activated microglia are believed to play different roles during inflammation that may either be neuroprotective or promote neurodegeneration. Here, we have investigated the effects of FFAs on microglial response to inflammatory stimuli. Our results indicate that the saturated FFA palmitate on its own induces alternative activation of BV-2 microglia cells. Further, pre-exposure to palmitate changed the response of microglia to lipopolysaccharide (LPS). We show that palmitate affects the mRNA levels of the pro-inflammatory cytokines interleukin-1β and interleukin-6. The transcription factor CCAAT/enhancer-binding protein δ is also affected by pre-exposure to palmitate. Furthermore, the phagocytic activity of microglia was investigated using fluorescent beads. By analyzing the bead uptake by fluorescence-activated cell sorting, we found that palmitate alone, as well as together with LPS, stimulated the phagocytic activity of microglia. Taken together, our results suggest that exposure of microglia to increased levels of free fatty acids may alter the consequences of classical inflammatory stimuli.

National Category
Biochemistry and Molecular Biology Neurosciences
Identifiers
urn:nbn:se:su:diva-95103 (URN)10.1007/s12031-013-0068-7 (DOI)000325710500021 ()
Available from: 2013-10-21 Created: 2013-10-21 Last updated: 2022-02-24Bibliographically approved
Gatsinzi, T., Ivanova, E. V. & Iverfeldt, K. (2012). TRAIL resistance in human neuroblastoma SK-N-AS cells is dependent on protein kinase C and involves inhibition of caspase-3 proteolytic processing. Journal of Neuro-Oncology, 109(3), 503-512
Open this publication in new window or tab >>TRAIL resistance in human neuroblastoma SK-N-AS cells is dependent on protein kinase C and involves inhibition of caspase-3 proteolytic processing
2012 (English)In: Journal of Neuro-Oncology, ISSN 0167-594X, E-ISSN 1573-7373, Vol. 109, no 3, p. 503-512Article in journal (Refereed) Published
Abstract [en]

Neuroblastoma is the most common solid extracranial cancer form in childhood with an etiology that is mostly unknown. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising future anticancer drug candidate, highly malignant neuroblastoma has been reported to acquire TRAIL resistance by mechanisms that are poorly understood. Here, we show by western blot analysis, and live cell imaging using anchored FRET sensors, that the resistance to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells depends on an incomplete processing of procaspase-3, generating an immature and catalytically inactive 21 kDa fragment. We have previously shown that the naturally occurring compound curcumin can sensitize SK-N-AS cells to TRAIL. In the present study, we show that curcumin also has a similar effect on human neuroblastoma SHEP1 cells. Furthermore, we show that curcumin and TRAIL co-treatment induces complete maturation and activation of caspase-3 in both cell lines. The mechanisms behind this effect seem to be dependent on protein kinase C (PKC), since inhibition of PKC using bisindolylmaleimide XI, could also sensitize these cells to TRAIL through a similar effect on caspase-3 activation. Moreover, TRAIL co-treatment with bisindolylmaleimide XI or curcumin resulted in down-regulation of X-linked inhibitor of apoptosis protein. In conclusion, our study shows that PKC can be involved in TRAIL resistance in human neuroblastoma cells by preventing caspase-3 maturation.

Keywords
AFS, Caspases, Curcumin, Neuroblastoma, PKC, TRAIL
National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-79146 (URN)10.1007/s11060-012-0932-2 (DOI)000308441700007 ()22798207 (PubMedID)
Funder
Swedish Research Council
Available from: 2012-08-28 Created: 2012-08-28 Last updated: 2022-02-24Bibliographically approved
Koistinen, N., Menon, P., Ivanova, E., Kumcu, M., Iverfeldt, K. & Ström, A.-L.ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 is attenuated in neuronally differentiated SH-SY5Y cells.
Open this publication in new window or tab >>ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 is attenuated in neuronally differentiated SH-SY5Y cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Fe65 is a brain enriched adaptor protein involved in various cellular processes. These processes may include regulated intramembrane proteolysis (RIP) of the amyloid-β precursor protein (APP) and transcriptional activation. However, much still needs to be learned regarding the regulation of Fe65 functions throughout the cell. In this study we therefore investigated the role of Fe65 Ser228 phosphorylation and α-secretase processing of proteins like APP undergoing RIP, in the regulation of Fe65 nuclear localization. We found that although Ser228 phosphorylation is not a major regulator of Fe65 nuclear localization, mutation of Ser228 results in an increased interaction with APP, suggesting that the N-terminal domain of Fe65 may have a more prominent role in mediating the Fe65-APP interaction than previously thought.  Moreover, we found that α-secretase processing play a key role in promoting Fe65 nuclear localization, but while ADAM10 play a considerable role in undifferentiated cells, other α-secretases take a more prominent part in releasing Fe65 from the plasma membrane in differentiated cells.      

Keywords
ADAM10, Fe65, APP
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-149905 (URN)
Available from: 2017-12-11 Created: 2017-12-11 Last updated: 2022-02-28Bibliographically approved
Tracy, L., Strååt, Y., Ivanova, E., Bergqvist, F. & Iverfeldt, K.Fibril-enriched amyloid-β inhibits interleukin-1 induced expression of the transcription factor C/EBPδ in astrocytes but not in microglia.
Open this publication in new window or tab >>Fibril-enriched amyloid-β inhibits interleukin-1 induced expression of the transcription factor C/EBPδ in astrocytes but not in microglia
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(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-95102 (URN)
Available from: 2013-10-21 Created: 2013-10-21 Last updated: 2022-02-24Bibliographically approved
Ivanova, E. V., Gatsinzi, T., Figueroa, R. A., Hallberg, E. & Iverfeldt, K.Increased spatiotemporal resolution of caspase activation by anchoring FRET-based sensors to cytoskeleton.
Open this publication in new window or tab >>Increased spatiotemporal resolution of caspase activation by anchoring FRET-based sensors to cytoskeleton
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(English)Manuscript (preprint) (Other academic)
Keywords
AFS, Apoptosis, Caspases, FRET, Live Cell Imaging
National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-79138 (URN)
Funder
Swedish Research Council
Available from: 2012-08-28 Created: 2012-08-28 Last updated: 2022-02-24Bibliographically approved
Menon, P., Koistinen, N., Ivanova, E., Cañellas Calafat, G., Revol, R., Iverfeldt, K. & Ström, A.-L.Mutation of Fe65-Ser228 enhances the Fe65-APP interaction and decreases non-amyloidogenic processing of APP.
Open this publication in new window or tab >>Mutation of Fe65-Ser228 enhances the Fe65-APP interaction and decreases non-amyloidogenic processing of APP
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Alzheimer’s disease is a neurodegenerative disease characterized by aberrant proteolysis of the transmembrane protein APP. The brain enriched adaptor protein Fe65 interacts with APP and participates together with APP and/or APP fragments in a number of cytoplasmic and nuclear functions. However, how the Fe65 subcellular localization, interaction with APP/APP fragments are regulated, as well as how Fe65 influences APP processing, is still not fully understood. In this study, we investigated the effect of Fe65 Ser-228 phosphorylation on Fe65 nuclear localization, APP interaction and APP processing. We show that although a Ser-228 phosphomimetic variant of Fe65 (Fe65-S2285E) was not excluded from the nucleus, a clear reduction of the nuclear level and the nuclear/cytoplasmic ratio of Fe65-S228E could be observed, suggesting that phosphorylation of Ser-288 could participate in regulation of the Fe65 subcellular localization. Interestingly, we found that not only Fe65-S2285E, but also mutation of Ser-228 to alanine (Fe65-S228A) resulted in a similar and dramatic increase of the Fe65 interaction with full-length APP. Moreover, we found that this increased APP interaction resulted in reduced α-secretase processing of APP and thus less generation of the neuroprotective sAPPα fragment. This suggest that the N-terminal domain of Fe65 may have a more prominent role in mediating the Fe65-APP interaction and regulating APP processing than previously thought.

Keywords
Alzheimer's disease, Fe65, amyloid precursor protein
National Category
Cell and Molecular Biology Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-181017 (URN)
Funder
Swedish Research Council, 521-2012-2367
Available from: 2020-04-24 Created: 2020-04-24 Last updated: 2022-02-26Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-1007-747x

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