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  • 1.
    Adlerz, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Beckman, Marie
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Holback, Sofia
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Tehranian, Roya
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Cortés Toro, Veronica
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Accumulation of the amyloid precursor-like protein APLP2 and reduction of APLP1 in retinoic acid-differentiated human neuroblastoma cells upon curcumin-induced neurite retraction2003In: Brain Research. Molecular Brain Research, ISSN 0169-328X, E-ISSN 1872-6941, Vol. 119, no 1, p. 62-72Article in journal (Refereed)
    Abstract [en]

    Amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. The function of these three proteins is not yet fully understood. One of the proposed roles of APP is to promote neurite outgrowth. The aim of this study was to investigate the regulation of the expression levels of APP family members during neurite outgrowth. We observed that retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y cells resulted in increased expression of APP, APLP1 and APLP2. We also examined the effect of the NFκB, AP-1 and c-Jun N-terminal kinase inhibitor curcumin (diferuloylmethane) on the RA-induced expression levels of these proteins. We found that treatment with curcumin counteracted the RA-induced mRNA expression of all APP family members. In addition, we observed that curcumin treatment resulted in neurite retraction without any effect on cell viability. Surprisingly, curcumin had differential effects on the APLP protein levels in RA-differentiated cells. RA-induced APLP1 protein expression was blocked by curcumin, while the APLP2 protein levels were further increased. APP protein levels were not affected by curcumin treatment. We propose that the sustained levels of APP and the elevated levels of APLP2, in spite of the reduced mRNA expression, are due to altered proteolytic processing of these proteins. Furthermore, our results suggest that APLP1 does not undergo the same type of regulated processing as APP and APLP2.

  • 2.
    Adlerz, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Holback, Sofia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Multhaup, Gerd
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    IGF-1-induced Processing of the Amyloid Precursor Protein Family Is Mediated by Different Signaling Pathways2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 14, p. 10203-10209Article in journal (Refereed)
    Abstract [en]

    The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.

  • 3.
    Adlerz, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Soomets, Ursel
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. University of Tartu, Estonia.
    Holmlund, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Virland, Saade
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Down-regulation of amyloid precursor protein by peptide nucleic acid oligomer in cultured rat primary neurons and astrocytes2003In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 336, no 1, p. 55-59Article in journal (Refereed)
    Abstract [en]

    The amyloid precursor protein (APP) and its proteolytic cleavage products, the amyloid P peptides, have been implicated as a cause of Alzheimer's disease. Peptide nucleic acids (PNA), the DNA mimics, have been shown to block the expression of specific proteins at both transcriptional and translational levels. Generally, the cellular uptake of PNA is low. However, recent studies have indicated that the effect of unmodified antisense PNA uptake is more pronounced in nervous tissue. In this study we have shown that biotinylated PNA directed to the initiator codon region of the APP mRNA (-4 - +11) was taken up into the cytoplasm of primary rat cerebellar granule cells and cortical astrocytes, using fluorescence and confocal microscopy studies. Uptake of PNA was faster in neurons than in astrocytes. Western blotting analysis showed that APP was strongly down-regulated in both neurons and astrocytes. Thus, unmodified PNA can be used for studies on the function of APP in neurons and astrocytes.

  • 4. Darsalia, Vladimer
    et al.
    Mansouri, Shiva
    Ortsater, Henrik
    Olverling, Anna
    Nozadze, Nino
    Kappe, Camilla
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Tracy, Linda M.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Grankvist, Nina
    Sjöholm, Åke
    Patrone, Cesare
    Glucagon-like peptide-1 receptor activation reduces ischaemic brain damage following stroke in Type 2 diabetic rats2012In: Clinical Science, ISSN 0143-5221, E-ISSN 1470-8736, Vol. 122, no 9-10, p. 473-483Article in journal (Refereed)
    Abstract [en]

    Diabetes is a strong risk factor for premature and severe stroke. The GLP-IR (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic Goto-Kakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 mu g/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 2-4 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-IR agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions.

  • 5.
    Figueroa, Ricardo A.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ramberg, Veronica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gatsinzi, Tom
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Samuelsson, Malin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zhang, Mu
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Anchored FRET sensors detect local caspase activation prior to neuronal degeneration2011In: Molecular Neurodegeneration, ISSN 1750-1326, E-ISSN 1750-1326, Vol. 6, p. 35-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-beta peptide as a causative agent in AD.

    RESULTS: Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-beta peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed.

    CONCLUSIONS: Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-beta peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.

  • 6.
    Fisher, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Samuelsson, Malin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ramberg, Veronica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo
    Södertörn University College, Sweden.
    Hallberg, Einar
    Södertörn University College, Sweden.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Targeting cytokine expression in glial cells by cellular delivery of an NFκB decoy2007In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 31, no 3, p. 209-219Article in journal (Refereed)
    Abstract [en]

    Inhibition of nuclear factor (NF)-κB has emerged as an important strategy for design of anti-inflammatory therapies. In neurodegenerative disorders like Alzheimer’s disease, inflammatory reactions mediated by glial cells are believed to promote disease progression. Here, we report that uptake of a double-stranded oligonucleotide NF-κB decoy in rat primary glial cells is clearly facilitated by noncovalent binding to a cell-penetrating peptide, transportan 10, via a complementary peptide nucleic acid (PNA) sequence. Fluorescently labeled oligonucleotide decoy was detected in the cells within 1 h only when cells were incubated with the decoy in the presence of cell-penetrating peptide. Cellular delivery of the decoy also inhibited effects induced by a neurotoxic fragment of the Alzheimer β amyloid peptide in the presence of the inflammatory cytokine interleukin (IL) 1β. Pretreatment of the cells with the complex formed by the decoy and the cell-penetrating peptide-PNA resulted in 80% and 50% inhibition of the NF-κB binding activity and IL-6 mRNA expression, respectively.

  • 7.
    Fisher, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Soomets, Ursel
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. University of Tartu, Estonia.
    Cortes Toro, Veronica
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Chilton, Lucy
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Langel, Ulo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Cellular delivery of a double-stranded oligonucleotide NFκB decoy by hybridization to complementary PNA linked to a cell-penetrating peptide2004In: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 11, p. 1264-1272Article in journal (Refereed)
    Abstract [en]

    The activation of nuclear factor B (NFB) is a key event in immune and inflammatory responses. In this study, a cell-penetrating transport peptide, transportan (TP) or its shorter analogue TP 10, was used to facilitate the cellular uptake of an NFB decoy. Peptide nucleic acid (PNA) hexamer or nonamer was linked to the transport peptide by a disulfide bond. NFB decoy oligonucleotide consisted of a double-stranded consensus sequence corresponding to the B site localized in the IL-6 gene promoter, 5'-GGGACTTTCCC-3', with a single-stranded protruding 3'-terminal sequence complementary to the PNA sequence was hybridized to the transport peptide–PNA construct. The ability of the transport peptide–PNA–NFB decoy complex to block the effect of interleukin (IL)-1-induced NFB activation and IL-6 gene expression was analyzed by electrophoretic mobility shift assay and reverse transcriptase-polymerase chain reaction in rat Rinm5F insulinoma cells. Preincubation with transport peptide–PNA–NFB decoy (1 M, 1 h) blocked IL-1-induced NFB-binding activity and significantly reduced the IL-6 mRNA expression. The same concentration of NFB decoy in the absence of transport peptide–PNA had no effect even after longer incubations. Our results showed that binding of the oligonucleotide NFB decoy to the nonamer PNA sequence resulted in a stable complex that was efficiently translocated across the plasma membrane.

  • 8.
    Gatsinzi, Tom
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ivanova, Elena V.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    TRAIL resistance in human neuroblastoma SK-N-AS cells is dependent on protein kinase C and involves inhibition of caspase-3 proteolytic processing2012In: Journal of Neuro-Oncology, ISSN 0167-594X, E-ISSN 1573-7373, Vol. 109, no 3, p. 503-512Article in journal (Refereed)
    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.

  • 9.
    Gatsinzi, Tom
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sensitization to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells by NF-kappa B inhibitors is dependent on reactive oxygen species (ROS)2011In: Journal of Neuro-Oncology, ISSN 0167-594X, E-ISSN 1573-7373, Vol. 104, no 2, p. 459-472Article in journal (Refereed)
    Abstract [en]

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in a variety of cancer cell lines with almost no toxicity toward normal cells. However, many neuroblastoma cells acquire resistance to TRAIL by mechanisms that are poorly understood. The objective of this study was to investigate involvement of the transcription factor NF-kappa B in the resistance of human neuroblastoma SK-N-AS cells to TRAIL-induced apoptosis. We used five compounds previously reported to inhibit NF-kappa B activity. SN50, curcumin, oridonin, and pyrrolidine dithiocarbamate (PDTC) all sensitized cells to TRAIL-induced apoptosis. In contrast, N-alpha-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) did not affect sensitivity to TRAIL, although reporter gene assay clearly showed inhibition of NF-kappa B activity. In addition, neither curcumin nor oridonin had any inhibitory effect on NF-kappa B activity at concentrations at which sensitization to TRAIL was observed. Instead, the free radical scavenger N-acetyl-l-cysteine (NAC) completely blocked the effect on TRAIL-induced apoptosis caused by curcumin, oridonin, and PDTC. Furthermore, exposure of SK-N-AS cells to H(2)O(2) could mimic the TRAIL-sensitizing effect of other agents. In conclusion, our results suggest that sensitization of neuroblastoma SK-N-AS cells to TRAIL-induced apoptosis is correlated with induction of reactive oxygen species (ROS) rather than inhibition of NF-kappa B.

  • 10.
    Gatsinzi, Tom
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Different strategies to inhibit NF-κB in order to sensitize Stypehuman neuroblastoma cells to TRAIL-induced apoptosisManuscript (preprint) (Other academic)
  • 11.
    Holback, Sofia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Adlerz, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gatsinzi, Tom
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The APP processing enzyme ADAM10 is up-regulated by retinoic acid in a PI3-kinase-dependent mannerIn: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468Article in journal (Refereed)
  • 12.
    Holback, Sofia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Adlerz, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gatsinzi, Tom
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jacobsen, Kristin T.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid2008In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 365, no 2, p. 298-303Article in journal (Refereed)
    Abstract [en]

    Retinoic acid stimulates α-secretase processing of amyloid precursor protein (APP) and decreases β-secretase cleavage that leads to amyloid-β formation. Here, we investigated the effect of retinoic acid on the two putative α-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the β-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of α-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.

  • 13.
    Holback, Sofia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Adlerz, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Increased processing of APLP2 and APP with concomitantformation of APP intracellular domains in BDNF and retinoic acid-differentiated human neuroblastoma cells2005In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 95, no 4, p. 1059-1068Article in journal (Refereed)
    Abstract [en]

    The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPalpha as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPalpha, as well as the levels of AICDs increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the alpha-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1.

  • 14.
    Holback, Sofia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Koistinen, Niina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jacobsen, Kristin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Retinoic acid stimulates maturation of the adaptor protein Fe65 and its binding to the amyloid precursor proteinManuscript (preprint) (Other academic)
    Abstract [en]

    Retinoic acid (RA) stimulates both synthesis and processing of the amyloid precursor protein (APP) and its mammalian paralogues, the APP-like proteins 1 and 2 (APLP1 and APLP2). Previously, we have detected increased levels of the APP and APLP1 intracellular dolmans, AICD and ALID1 respectively, concomitant with RA-induced neuronal differentiation. Here we used Western blot analysis to show increased levels of the mature form of the adaptor protein Fe65 during RA- as well as nerve growth factor-induced differentiation. Co-immunoprecipitation studies also revealed that increased binding of Fe65 to APP and APLP1 occurred during neuronal differentiation. Furthermore, exposure to RA decreased the phosphorylation of Thr668 located in the cytoplasmic domain of APP.

  • 15.
    Ivanova, Elena V.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo A.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gatsinzi, Tom
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution2016In: Sensors, ISSN 1424-8220, E-ISSN 1424-8220, Vol. 16, no 5, article id 703Article in journal (Refereed)
    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.

  • 16.
    Ivanova, Elena V.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gatsinzi, Tom
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo A.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Increased spatiotemporal resolution of caspase activation by anchoring FRET-based sensors to cytoskeletonManuscript (preprint) (Other academic)
  • 17.
    Jacobsen, Kristin
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Adlerz, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Multhaup, Gerd
    Institute for chemistry and biochemistry, Free University of Berlin.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Insulin-like growth factor-1 (IGF-1)-induced processing of amyloid-β precursor protein (APP) and APP-like protein 2 is mediated by different metalloproteinases2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 14, p. 10223-10231Article in journal (Refereed)
    Abstract [en]

    α-Secretase cleavage of the amyloid precursor protein (APP) is of great interest since it prevents the formation of the Alzheimer-linked amyloid-β peptide. APP belongs to a conserved gene family including the two paralogues APP-like protein (APLP) 1 and 2. Insulin-like growth factor-1 (IGF-1) stimulates the shedding of all three proteins. IGF-1-induced shedding of both APP and APLP1 is dependent on phosphatidylinositol 3-kinase (PI3-K), whereas sAPLP2 secretion is independent of this signaling pathway. Here, we used human neuroblastoma SH-SY5Y cells to investigate the involvement of protein kinase C (PKC) in the proteolytic processing of endogenously expressed members of the APP family. Processing was induced by IGF-1 or retinoic acid, another known stimulator of APP a-secretase shedding. Our results show that stimulation of APP and APLP1 processing involves multiple signaling pathways, whereas APLP2 processing is mainly dependent on PKC. Next, we wanted to investigate if the difference in the regulation of APLP2 shedding compared to APP shedding could be due to involvement of different processing enzymes. We focused on the two major a-secretase candidates ADAM10 and TACE, which both are members of the ADAM (a disintegrin and metalloprotease) family. Shedding was analyzed in the presence of the ADAM10 inhibitor GI254023X, or after transfection with siRNA targeted against TACE. The results clearly demonstrate that different α-secretases are involved in IGF-1-induced processing. APP is mainly cleaved by ADAM10, whereas APLP2 processing is mediated by TACE. Finally, we also show that IGF-1 induces PKC-dependent phosphorylation of TACE.

  • 18.
    Jacobsen, Kristin T
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Amyloid precursor protein and its homologues: a family of proteolysis-dependent receptors.2009In: Cellular and molecular life sciences : CMLS, ISSN 1420-9071, Vol. 66, no 14, p. 2299-2318Article in journal (Refereed)
    Abstract [en]

    The Alzheimer's amyloid precursor protein (APP) belongs to a conserved gene family that also includes the mammalian APLP1 and APLP2, the Drosophila APPL, and the C. elegans APL-1. The biological function of APP is still not fully clear. However, it is known that the APP family proteins have redundant and partly overlapping functions, which demonstrates the importance of studying all APP family members to gain a more complete picture. When APP was first cloned, it was speculated that it could function as a receptor. This theory has been further substantiated by studies showing that APP and its homologues bind both extracellular ligands and intracellular adaptor proteins. The APP family proteins undergo regulated intramembrane proteolysis (RIP), generating secreted and cytoplasmic fragments that have been ascribed different functions. In this review, we will discuss the APP family with focus on biological functions, binding partners, and regulated processing.

  • 19.
    Jacobsen, Kristin T.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-beta precursor protein (APP)2011In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 404, no 3, p. 882-886Article in journal (Refereed)
    Abstract [en]

    The amyloid-beta precursor protein (APP) was shown to be O-GlcNAcylated 15 years ago, but the effect of this modification on APP processing and formation of the Alzheimer's disease associated amyloid-beta (A beta) peptide has so far not been investigated. Here, we demonstrate with pharmacological tools or siRNA that O-GlcNAcase and O-GlcNAc transferase regulate the level of O-GlcNAcylated APP. We also show that O-GlcNAcylation increases non-amyloidogenic alpha-secretase processing, resulting in increased levels of the neuroprotective sAPP alpha fragment and decreased A beta secretion. Our results implicate O-GlcNAcylation as a potential therapeutic target for Alzheimer's disease.

  • 20.
    Jacobsen, Kristin T.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The E1 domain of APP and APLP2 determines α-secretase specificityManuscript (preprint) (Other academic)
    Abstract [en]

    The α-secretase cleavage of the amyloid-β precursor protein (APP) precludes the formation of amyloid-β (Aβ), the main constituent of senile plaques in Alzheimer´s disease (AD). Stimulation of α-secretase processing may thereby constitute an important therapeutical strategy. APP belongs to a conserved protein family including the APP-like protein 2 (APLP2). Although the proteins are sequentially processed in a similar way, we have previously shown that insulin-like factor-1 (IGF-1) - and retinoic acid (RA)-induced α-secretase processing of APP and APLP2 is mediated by different enzymes. APP was shown to be cleaved by the α-secretase ADAM10 in a PI3K-dependent manner, whereas APLP2 was cleaved by the α-secretase TACE in a PKC-dependent manner. To better understand the mechanism underlying this difference in α-secretase processing between these two homologous proteins, we aimed to determine which part of APP that was essential for making it a better substrate for ADAM10 than for TACE during stimulated conditions. We constructed a chimeric protein, were the E1 domain of APP was substituted with the corresponding domain of APLP2. Our results demonstrate that the APP/E1/APLP2 chimer is successfully expressed and secreted into the cell medium of transiently transfected SH-SY5Y cells. Pharmacological inhibition demonstrate that the IGF-1-induced processing of the chimer is PI3K-independent but dependent on PKC, as previously shown for the APLP2 processing. Our result indicates that the E1 domain in APP determines its specificity towards ADAM10 over TACE.

  • 21.
    Jacobsen, Kristin T.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Strååt, Ylva
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Koistinen, Niina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    O-GlcNAcylation of the α-secretase ADAM10 selectively affects APP processing in neuron-like cellsManuscript (preprint) (Other academic)
    Abstract [en]

    α-Secretase processing of APP has recently gained more interest, highlighting its potential as a therapeutic target to prevent Alzheimer’s disease (AD). We have previously shown that O-GlcNAcylation stimulates α-secretase processing of APP, concomitantly with decreased Aβ secretion. O-GlcNAcylation has previously been linked to AD since the levels of O-GlcNAcylated proteins are decreased in AD brains. Here, we have further investigated the mechanism behind α-secretase processing of APP in response to increased O-GlcNAcylation. Our results shown that APP is not O-GlcNAcylated during the conditions used in this study. Instead, we demonstrate that the α-secretase ADAM10 is O-GlcNAcylated and that APP cell surface localization is enhanced in response to increased O-GlcNAcylation. Furthermore, the effects of O-GlcNAcylation on APP processing are cell-type specific, only affecting sAPPα secretion in neuroblastoma cell-lines.

  • 22. Kappe, Camilla
    et al.
    Tracy, Linda M.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Patrone, Cesare
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sjöholm, Åke
    GLP 1 secretion by microglial cells and decreased cns expression in obesity2012In: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 9, p. 276-Article in journal (Refereed)
    Abstract [en]

    Background: Type 2 diabetes (T2D) is a strong risk factor for developing neurodegenerative pathologies. T2D patients have a deficiency in the intestinal incretin hormone GLP-1, which has been shown to exert neuroprotective and anti-inflammatory properties in the brain. Methods: Here we investigate potential sources of GLP-1 in the CNS and the effect of diabetic conditions on the proglucagon mRNA expression in the CNS. The obese mouse model ob/ob, characterized by its high levels of free fatty acids, and the microglia cell line BV-2 were used as models. mRNA expression and protein secretion were analyzed by qPCR, immunofluorescence and ELISA. Results: We show evidence for microglia as a central source of GLP-1 secretion. Furthermore, we observed that expression and secretion are stimulated by cAMP and dependent on microglial activation state. We also show that insulin-resistant conditions reduce the central mRNA expression of proglucagon. Conclusion: The findings that microglial mRNA expression of proglucagon and GLP-1 protein expression are affected by high levels of free fatty acids and that both mRNA expression levels of proglucagon and secretion levels of GLP-1 are affected by inflammatory stimuli could be of pathogenic importance for the premature neurodegeneration and cognitive decline commonly seen in T2D patients, and they may also be harnessed to advantage in therapeutic efforts to prevent or treat such disorders.

  • 23.
    Kihlmark, Madeleine
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Södertörn University College, Sweden.
    Rustum, Cecilia
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Södertörn University College, Sweden.
    Eriksson, Charlotta
    Beckman, Marie
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Hallberg, Einar
    Correlation between nucleocytoplasmic transport and caspase–3-dependent dismantling of nuclear pores during apoptosis2004In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 293, no 2, p. 346-356Article in journal (Refereed)
    Abstract [en]

    During apoptosis (also called programmed cell death), the chromatin condenses and the DNA is cleaved into oligonucleosomal fragments. Caspases are believed to play a major role in nuclear apoptosis. However, the relation between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. We have analyzed nuclear import of the green fluorescent protein fused to a nuclear localization signal (GFP-NLS) in tissue culture cells undergoing apoptosis. Decreased nuclear accumulation of GFP-NLS could be detected at the onset of nuclear apoptosis manifested as dramatic condensation and redistribution of chromatin toward the nuclear periphery. At this step, dismantling of nuclear pores was already evident as indicated by proteolysis of the nuclear pore membrane protein POM121. Thus, disruption of nuclear compartmentalization correlated with early signs of nuclear pore damage. Both these events clearly preceded massive DNA fragmentation, detected by TUNEL assay. Furthermore, we show that in apoptotic cells, POM121 is specifically cleaved at aspartate-531 in its large C-terminal portion by a caspase-3-dependent mechanism. Cleavage of the C-terminal portion of POM121, which is adjoining the nuclear pore complex, is likely to disrupt interactions with other nuclear pore proteins affecting the stability of the pore complex. A temporal correlation of apoptotic events supports a model where caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation.

  • 24.
    Koistinen, Niina A.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Bacanu, Smaranda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Phosphorylation of Fe65 amyloid precursor protein-binding protein in response to neuronal differentiation2016In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 613, p. 54-59Article in journal (Refereed)
    Abstract [en]

    Fe65 is a brain enriched multi domain adaptor protein involved in diverse cellular functions. One of its binding partners is the amyloid-beta (A beta) precursor protein (APP), which after sequential proteolytic processing by secretases gives rise to the Alzheimer's A beta peptide. Fe65 binds to the APP intracellular domain (AICD). Several studies have indicated that Fe65 binding promotes the amyloidogenic processing of APP. It has previously been shown that expression of APP increases concomitantly with a shift of its processing to the non-amyloidogenic pathway during neuronal differentiation. In this study we wanted to investigate the effects of neuronal differentiation on Fe65 expression. We observed that differentiation of SH-SY5Y human neuroblastoma cells induced by retinoic acid (RA), the phorbol ester PMA, or the gamma-secretase inhibitor DAPT resulted in an electrophoretic mobility shift of Fe65. Similar effects were observed in rat PC6.3 cells treated with nerve growth factor. The electrophoretic mobility shift was shown to be due to phosphorylation. Previous studies have shown that Fe65 phosphorylation can prevent the APP-Fe65 interaction. We propose that phosphorylation is a way to modify the functions of Fe65 and to promote the non-amyloidogenic processing of APP during neuronal differentiation.

  • 25.
    Koistinen, Niina A.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Edlund, Anna K.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Menon, Preeti K.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ivanova, Elena V.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Bacanu, Smaranda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Nuclear localization of amyloid-beta precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 3, article id e0173888Article in journal (Refereed)
    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.

  • 26.
    Koistinen, Niina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Holback, Sofia
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    siRNA knock-down of Fe65 in SH-SY5Y cells decreases the levels of C-terminal fragments of APP without any effect on sAPPα secretionManuscript (preprint) (Other academic)
    Abstract [en]

    Fe65 is an adaptor protein that binds to the amyloid precursor protein (APP) within the 82YENPTY687 motif of APP which is important for amyloid β (Aβ) production. Considering that Fe65 binds to this motif, it can be hypothesized that Fe65 may influence the trafficking of APP and hence its processing by α- and/or β-secretase. Therefore in this study we wanted to determine how knock-down of Fe65 effects the processing of endogenous APP in human SH-SY5Y neuroblastoma cells. Our results showed that Fe65 knock-down did not have any effect on sAPPα secretion in SH-SY5Y cells. However, decreased levels of membrane-bound APP stubs C83 and C99 were observed, suggesting that Fe65 has a stabilizing effect on the C-terminal fragments. Furthermore, we wanted to investigate effects of retinoic acid (RA)-induced neronal differentiation on Fe65 expression. It has previously been shown that under these conditions mRNA and protein levels of APP increase concomitant with increased secretion of sAPPα, shifting the processing of APP to the more non-amyloidogenic pathway. We observed that RA-induced neuronal differentiation increases the protein levels of Fe65 in SH-SY5Y cells and gives rise to an electrophoretic mobility shift due to increased phosphorylation. The increased expression levels of Fe65 during neuronal differentiation concomitant with the increase of Fe65 phosphorylation, suggest that Fe65 and its phosphorylation may play a role during neuronal differentiation.

  • 27.
    Koistinen, Niina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Menon, Preeti
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ivanova, Elena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kumcu, Michael
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ström, Anna-Lena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 is attenuated in neuronally differentiated SH-SY5Y cellsManuscript (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.      

  • 28.
    Koistinen, Niina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Menon, Preeti
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ström, Anna-Lena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    APP Ser675 phosphorylation affects α-secretase processing resulting in decreased secretion of the neuroprotective ectodomain sAPPαManuscript (preprint) (Other academic)
    Abstract [en]

    Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of the amyloid-β (Aβ) peptide. Aβ is produced after amyloidogenic (β-secretase) processing of the transmembrane amyloid precursor protein (APP). However, APP can also be processed by α-secretases, instead resulting in release of neuroprotective sAPPα.  Growing evidence indicate that aberrant post-translational modifications of APP may play a pivotal role in AD pathogenesis by dysregulating APP processing. APP Ser675 phosphorylation occurs in AD brains and here we for the first time show that this phosphorylation decreases the release of sAPPα, while the level of an alternative APP-C83-CTF fragment is increased. Moreover, we found that while APP Ser675 phosphorylation increased the APP-Fe65 interaction, the level of APP at the plasma membrane were unaltered. Taken together these results suggest that APP Ser675 phosphorylation alters the α-secretase processing of APP at the plasma membrane. As α-secretase processing of APP is an essential step in decreasing the generation of Aβ these results suggest that Ser675 phosphorylation could contribute to AD pathology.

  • 29.
    Ramberg, Veronica
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Tracy, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Samuelsson, Malin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Nilsson, Lars N.G.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The CCAAT/enhancer binding protein (C/EBP) δ is differently regulated by fibrillar and oligomeric forms of the Alzheimer amyloid-β peptide2011In: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 8, p. 34-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    The transcription factors CCAAT/enhancer binding proteins (C/EBP) α, β and δ have been shown to be expressed in brain and to be involved in regulation of inflammatory genes in concert with nuclear factor κB (NF-κB). In general, C/EBPα is down-regulated, whereas both C/EBPβ and δ are up-regulated in response to inflammatory stimuli. In Alzheimer's disease (AD) one of the hallmarks is chronic neuroinflammation mediated by astrocytes and microglial cells, most likely induced by the formation of amyloid-β (Aβ) deposits. The inflammatory response in AD has been ascribed both beneficial and detrimental roles. It is therefore important to delineate the inflammatory mediators and signaling pathways affected by Aβ deposits with the aim of defining new therapeutic targets.

    METHODS:

    Here we have investigated the effects of Aβ on expression of C/EBP family members with a focus on C/EBPδ in rat primary astro-microglial cultures and in a transgenic mouse model with high levels of fibrillar Aβ deposits (tg-ArcSwe) by western blot analysis. Effects on DNA binding activity were analyzed by electrophoretic mobility shift assay. Cross-talk between C/EBPδ and NF-κB was investigated by analyzing binding to a κB site using a biotin streptavidin-agarose pull-down assay.

    RESULTS:

    We show that exposure to fibril-enriched, but not oligomer-enriched, preparations of Aβ inhibit up-regulation of C/EBPδ expression in interleukin-1β-activated glial cultures. Furthermore, we observed that, in aged transgenic mice, C/EBPα was significantly down-regulated and C/EBPβ was significantly up-regulated. C/EBPδ, on the other hand, was selectively down-regulated in the forebrain, a part of the brain showing high levels of fibrillar Aβ deposits. In contrast, no difference in expression levels of C/EBPδ between wild type and transgenic mice was detected in the relatively spared hindbrain. Finally, we show that interleukin-1β-induced C/EBPδ DNA binding activity to both C/EBP and κB sites is abolished after exposure to Aβ.

    CONCLUSIONS:

    These data suggest that both expression and function of C/EBPδ are dysregulated in Alzheimer's disease. C/EBPδ seems to be differently regulated in response to different conformations of Aβ. We propose that Aβ induces an imbalance between NF-κB and C/EBP transcription factors that may result in abnormal responses to inflammatory stimuli.

  • 30.
    Samuelsson, Malin
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Fisher, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    β-Amyloid and interleukin-1β induce persistent NF-κB activation in rat primary glial cells2005In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 16, no 3, p. 449-453Article in journal (Refereed)
    Abstract [en]

    An increasing body of evidence suggests that β-amyloid (Aβ) and activated glial cells play a crucial part in the pathogenesis of Alzheimer's disease (AD). Activated glial cells surrounding the senile plaques, formed by Aβ peptides, have been proposed to promote neurodegeneration by producing putatively toxic factors, including the inflammatory cytokine interleukin-1β (IL-1β). Elevated levels of both IL-1β and activated nuclear factor κB (NF-κB), a key transcription factor regulating a wide variety of inflammatory genes, have been found in the brains of AD patients. In this study, we have investigated the ability of the Aβ(25-35) peptide and IL-1β, either alone or together, in activating NF-κB in glial cells. Mixed primary glial cells from rat were treated with IL-1β and/or Aβ(25-35), and NF-κB binding activity was analyzed by electophoretic mobility shift assay. We observed that the induction of NF-κB binding activity induced by either IL-1β or Aβ(25-35) showed a peak at 30 min, and significantly declined after 2 h. The induced NF-κB activation persisted after 24 h and even seemed to increase in cells treated with Aβ(25-35). The activation of NF-κB by Aβ(25-35) was shown to be dose-dependent. In addition, Aβ(25-35) potentiated the effect of IL-1β in a dose-dependent manner when co-stimulating the cells. The potentiating effect of Aβ(25-35) on IL-1β-induced NF-κB binding activity was observed after 30 min, 2 h and 24 h, and did not significantly differ over time. A possible explanation is that when glial cells are stimulated by inflammatory factors in the presence of Aβ peptides or senile plaques, the NF-κB negative feedback regulation is no longer functional.

  • 31.
    Samuelsson, Malin
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Alzheimer Aβ peptides induce кB binding complexes containing C/EBPδManuscript (Other academic)
  • 32.
    Samuelsson, Malin
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ramberg, Veronica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Alzheimer amyloid-beta peptides block the activation of C/EBP beta and C/EBP delta in glial cells2008In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 370, no 4, p. 619-622Article in journal (Refereed)
    Abstract [en]

    Members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors have been reported to be up-regulated in Alzheimer's disease. In the present study, we have investigated the effects of amyloid-beta (A beta) peptides on C/EBP beta and C/EBP delta, previously shown to be induced by inflammatory stimuli in glial cells. Surprisingly, electrophoretic mobility shift assay showed that both A beta(1-42) and A beta(25-35) blocked C/EBP activation induced by the inflammatory cytokine interleukin-1 (IL-1 beta) or lipopolysac-charicle (LPS) in mixed primary glial cell cultures from rat. A beta also blocked IL-1 or LPS-incluced C/EBP protein levels. The most prominent effects were observed on DNA binding activity and protein levels of C/EBP delta. Our results demonstrate a dysregulation of C/EBP when glial cells are activated in the presence of Alzheimer A beta peptides.

  • 33.
    Tracy, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Bergqvist, Filip
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ivanova, Elena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jacobsen, Kristin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Exposure to the Saturated Free Fatty Acid Palmitate AltersBV-2 Microglia Inflammatory Response2013In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 51, no 3, p. 805-812Article in journal (Refereed)
    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.

  • 34.
    Tracy, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Strååt, Ylva
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ivanova, Elena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Bergqvist, Filip
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Fibril-enriched amyloid-β inhibits interleukin-1 induced expression of the transcription factor C/EBPδ in astrocytes but not in microgliaManuscript (preprint) (Other academic)
1 - 34 of 34
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