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  • 151.
    Galofré, Mireia
    et al.
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona, and CIBER Epidemiología y Salud Pública (CIBERESP), Spain.
    Babot, Zoila
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona, and CIBER Epidemiología y Salud Pública (CIBERESP), Spain.
    García, Daniel A
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona,.
    Iraola, Susana
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona,.
    Rodríguez-Farré, Eduard
    Department of Brain Ischemia and Neurodegeneration, IIBB, CSIC-IDIBAPS, Spain and CIBER Epidemiología y Salud Pública (CIBERESP), Spain.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Suñol, Cristina
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona, and CIBER Epidemiología y Salud Pública (CIBERESP), Spain.
    GABA(A) receptor and cell membrane potential as functional endpoints in cultured neurons to evaluate chemicals for human acute toxicity2010Inngår i: Neurotoxicology and Teratology, ISSN 0892-0362, E-ISSN 1872-9738, Vol. 32, s. 52-61Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Toxicity risk assessment for chemical-induced human health hazards relies mainly on data obtained from animal experimentation, human studies and epidemiology. In vitro testing for acute toxicity based on cytotoxicity assays predicts 70 - 80% of rodent and human toxicity. The nervous system is particularly vulnerable to chemical exposure which may result in different toxicity features. Acute human toxicity related to adverse neuronal function is usually a result of over-excitation or depression of the nervous system. The major molecular and cellular mechanisms involved in such reactions include GABAergic, glutamatergic and cholinergic neurotransmission, regulation of cell and mitochondrial membrane potential, and those critical for maintaining central nervous system functionality, such as controlling cell energy. In this work, a set of chemicals that are used in pharmacy, industry, biocide treatments or are often abused by drug users are tested for their effects on GABA(A) receptor activity, GABA and glutamate transport, cell membrane potential and cell viability in primary neuronal cultures. GABA(A) receptor function was inhibited by compounds for which seizures have been observed after severe human poisoning. Commonly abused drugs inhibit GABA uptake but not glutamate uptake. Most neurotoxins altered membrane potential. The GABA(A) receptor, GABA uptake and cell membrane potential assays were those that identified the highest number of chemicals as toxic at low concentrations. These results show that in vitro cell assays may identify compounds that produce acute neurotoxicity in humans, provided that in vitro models expressing neuronal targets relevant for acute neural dysfunctions are used.

  • 152. Ganyu, Anita
    et al.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Good, Liam
    Microbial membrane-penetrating peptides and their application2007Inngår i: Handbook of cell-penetrating peptides / [ed] Ülo Langel, Boca Raton: CRC Press, 2007, 2, s. 515-533Kapittel i bok, del av antologi (Fagfellevurdert)
  • 153. Garcia-Sosa, Alfonso T.
    et al.
    Tulp, Indrek
    Langel, Kent
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Peptide-Ligand Binding Modeling of siRNA with Cell-Penetrating Peptides2014Inngår i: BioMed Research International, ISSN 2314-6133, E-ISSN 2314-6141, s. 257040-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The binding affinity of a series of cell-penetrating peptides (CPP) was modeled through docking and making use of the number of intermolecular hydrogen bonds, lipophilic contacts, and the number of sp3 molecular orbital hybridization carbons. The new ranking of the peptides is consistent with the experimentally determined efficiency in the downregulation of luciferase activity, which includes the peptides' ability to bind and deliver the siRNA into the cell. The predicted structures of the complexes of peptides to siRNA were stable throughout 10 ns long, explicit water molecular dynamics simulations. The stability and binding affinity of peptide-siRNA complexes was related to the sidechains and modifications of the CPPs, with the stearyl and quinoline groups improving affinity and stability. The reranking of the peptides docked to siRNA, together with explicit water molecular dynamics simulations, appears to be well suited to describe and predict the interaction of CPPs with siRNA.

  • 154.
    Gatsinzi, Tom
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Neuroblastoma cells: in vitro studies on childhood cancer and amyloid precursor protein processing enzymes2009Licentiatavhandling, med artikler (Annet vitenskapelig)
  • 155.
    Gatsinzi, Tom
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Regulation of apoptotic processes in neurodegeneration and cancer: In vitro studies on human neuroblastoma cells2012Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Apoptosis is a highly controlled process of cell death, which is vital for maintenance of all multicellular organisms. Aberrant regulation of apoptosis can give rise to pathological conditions such as neurodegenerative diseases and cancer. Here, we used different human neuroblastoma cell lines to study mechanisms that may be involved in either neurodegeneration or resistance to cancer treatment. First, we have designed and developed tau-anchored FRET sensors (tAFSs) for live cell imaging of local caspase activation. Using these sensors we showed that the Alzheimer’s disease related neurotoxic peptide, amyloid-β, induced a global activation of caspase-3 and -6, but not -9, in neuronally differentiated SH-SY5Y cells. We also investigated the possible role of NF-κB in the resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in different human neuroblastoma cell lines. While N-type SH-SY5Y and IMR32 cells were unaffected, S-type SK-N-AS cells were clearly sensitized to TRAIL by different NF-κB inhibitory agents. However, no correlation between NF-κB inhibition and sensitization to TRAIL could be observed. Instead, induction of reactive oxygen species (ROS) seemed to play a more important role. Furthermore, using tAFSs we also showed that TRAIL resistance in SK-N-AS cells is mainly due to incomplete activation of caspase-3, and could be reversed by different PKC inhibitors.

  • 156.
    Gatsinzi, Tom
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ivanova, Elena V.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    TRAIL resistance in human neuroblastoma SK-N-AS cells is dependent on protein kinase C and involves inhibition of caspase-3 proteolytic processing2012Inngår i: Journal of Neuro-Oncology, ISSN 0167-594X, E-ISSN 1573-7373, Vol. 109, nr 3, s. 503-512Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 157.
    Gatsinzi, Tom
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sensitization to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells by NF-kappa B inhibitors is dependent on reactive oxygen species (ROS)2011Inngår i: Journal of Neuro-Oncology, ISSN 0167-594X, E-ISSN 1573-7373, Vol. 104, nr 2, s. 459-472Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 158.
    Gatsinzi, Tom
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jiang, Yang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Different strategies to inhibit NF-κB in order to sensitize Stypehuman neuroblastoma cells to TRAIL-induced apoptosisManuskript (preprint) (Annet vitenskapelig)
  • 159. Govindarajan, Sridhar
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Silverman, Joshua A.
    Wright, Kathy
    Regitsky, Drew
    Hegazy, Usama
    Purcell, Thomas J.
    Welch, Mark
    Minshull, Jeremy
    Gustafsson, Claes
    Mapping of Amino Acid Substitutions Conferring Herbicide Resistance in Wheat Glutathione Transferase2015Inngår i: ACS Synthetic Biology, ISSN 2161-5063, Vol. 4, nr 3, s. 221-227Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have used design of experiments (DOE) and systematic variance to efficiently explore glutathione transferase substrate specificities caused by amino acid substitutions. Amino acid substitutions selected using phylogenetic analysis were synthetically combined using a DOE design to create an information-rich set of gene variants, termed infologs. We used machine learning to identify and quantify protein sequence-function relationships against 14 different substrates The resulting models were quantitative and predictive, serving as a guide for engineering of glutathione transferase activity toward a diverse set of herbicides Predictive quantitative models like those presented here have broad applicability for bioengineering.

  • 160. Groves-Chapman, Jessica L.
    et al.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holmes, Philip V.
    Galanin receptor agonists protect against kainic acid-induced excitotoxicity in the rat hippocampus2011Konferansepaper (Fagfellevurdert)
    Abstract [en]

    Galanin is a peptide neurotransmitter with neuroprotective actions. Administration of galanin or selective galanin receptor agonists to rats reduces convulsant-induced seizure behavior. However, it remains unclear whether galanin exerts a neuroprotective effect in vivo and which galanin receptor subtype may mediate this effect. The present experiments evaluated the impact of two separate galanin receptor agonists, M1145 and M1154, which are specific for GAL R2 and GAL R1/R2 respectively, on seizure behavior and neuronal excitotoxicity following intracerebroventricular (ICV) administration of kainic acid. Male, Sprague-Dawley rats received ICV infusions of M1145, M1154, or saline prior to an ICV infusion of kainic acid. Seizure behaviors were rated for 30 minutes post-infusion, and rats were euthanized and transcardially perfused 48 hours later. Twenty micron coronal sections of the hippocampal region were obtained and nissl-stained for microscopic cell counting of pyramidal neurons in the CA3 region.Neither galanin receptor agonist reduced the seizure behaviors induced by kainic acid. Cell counting in CA3 revealed that kainic acid treatment alone caused near complete destruction of cells. However, rats pretreated with the galanin receptor agonists showed reduced cell loss. These results provide further evidence of the protective role of galanin in the hippocampus and suggest that the GAL R2 receptor mediates these effects.

  • 161.
    Gudise, Santhosh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Karolinska Institute (NOVUM), Sweden.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindberg, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Larsson, Veronica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Samp1 is functionally associated with the LINC complex and A-type lamina networks2011Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 124, s. 2077-2085Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The transmembrane inner nuclear membrane (INM) protein Samp1 is required for anchoring centrosomes near the nuclei. Using high-resolution fluorescence microscopy we show that Samp1 is distributed in a distinct and characteristic pattern in the nuclear envelope (NE), where it partially colocalizes with the LINC complex protein Sun1. By studying the localization of Samp1 deletion mutants and fusion proteins, we conclude that the cysteine-rich N-terminal half of Samp1 is nucleoplasmically exposed and is responsible for targeting to the INM. It contains four conserved CxxC motifs with the potential to form zinc fingers. The distribution of cysteine-to-alanine substitution mutants, designed to prevent zinc finger formation, showed that NE localization of Samp1 depends on intact CxxC motifs. Overexpression of Samp1 zinc finger mutants produced an abnormal dominant phenotype characterized by disrupted organization of a selective subset NE proteins, including emerin, Sun1, endogenous Samp1 and, in some cases, lamin A/C, but not lamin B, Sun2 or nucleoporins. Silencing of Samp1 expression showed that emerin depends on Samp1 for its correct localization in the NE. Our results demonstrate that Samp1 is functionally associated with the LINC complex protein Sun1 and proteins of the A-type lamina network.

  • 162. Guipponi, Michel
    et al.
    Chentouf, Amina
    Webling, Kristin E. B.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Freimann, Krista
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Crespel, Arielle
    Nobile, Carlo
    Lemke, Johannes R.
    Hansen, Jörg
    Dorn, Thomas
    Lesca, Gaetan
    Ryvlin, Philippe
    Hirsch, Edouard
    Rudolf, Gabrielle
    Rosenberg, Dominique Sarah
    Weber, Yvonne
    Becker, Felicitas
    Helbig, Ingo
    Muhle, Hiltrud
    Salzmann, Annick
    Chaouch, Malika
    Oubaiche, Mohand Laid
    Ziglio, Serena
    Gehrig, Corinne
    Santoni, Federico
    Pizzato, Massimo
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Antonarakis, Stylianos E.
    Galanin pathogenic mutations in temporal lobe epilepsy2015Inngår i: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 24, nr 11, s. 3082-3091Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatment.

  • 163.
    Gustafsson, Helena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Uncoupling Proteins: Regulation by IGF-1 and Neuroprotection during Hyperglycemia in Vitro2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Diabetic neuropathy is believed to arise due to oxidative stress following hyperglycemic situations. Uncoupling proteins (UCPs) constitute a subgroup of mitochondrial transporter proteins with putative antioxidant properties. By dissipating the proton gradient over the mitochondrial inner membrane, these proteins reduce the mitochondrial inner membrane potential (MMP), and thereby, the mitochondrial production of reactive oxygen species (ROS) is decreased. In this thesis I have examined the regulation of UCP2, UCP3, and UCP4 by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1). I have also investigated the possible involvement of UCP3 in IGF-1-mediated neuroprotection following high glucose treatments. All studies were performed using human neuroblastoma SH-SY5Y cells as an in vitro cell model. The major findings were as follows:

    i. Native SH-SY5Y cells expressed UCP2, UCP3, and UCP4.

    ii. UCP3 was upregulated by IGF-1 via activation of the IGF-1 receptor. IGF-1 increased UCP3 mRNA and protein levels primarily via activation of the “classical” anti-apoptotic phosphatidyl inositol 3 (PI3)-kinase signaling pathway, as shown by incubation with specific inhibitors of the PI3-kinase and mitogen activated protein (MAP) kinase signaling pathways.

    iii. UCP2 and UCP4 protein levels were only marginally or not at all regulated by IGF-1. These UCPs are probably not involved in IGF-1-mediated neuroprotection.

    iv. High glucose concentrations reduced the UCP3 protein levels in highly differentiated SH-SY5Y cells. Concomitantly, the MMP and the levels of ROS and glutathione increased, whereas the number of neurites per cell was reduced. This supports an antioxidant and neuroprotective role of UCP3

    v. IGF-1 prevented the glucose-induced reduction in UCP3 protein levels. In parallel, the effects on MMP, levels of ROS and glutathione, and number of neurites per cell were abolished or significantly reduced. These data suggest that UCP3 is involved in IGF-1-mediated neuroprotection.

  • 164.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Adamson, Lars
    Hedander, Jan
    Walum, Erik
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin-like growth factor type 1 upregulates uncoupling protein 3.2001Inngår i: Biochem Biophys Res Commun, ISSN 0006-291X, Vol. 287, nr 5, s. 1105-11Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor. Copyright 2001 Academic Press.

  • 165.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Adamson, Lars
    Hedander, Jan
    Walum, Erik
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin-like growth factor type 1 up-regulates uncoupling protein 32001Inngår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 287, nr 5, s. 1105-1111Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor.

  • 166.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Expression of uncoupling protein 2 and 4 in human neuroblastoma SH-SY5Y cellsManuskript (Annet vitenskapelig)
  • 167.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindegren, Heléne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Axelsson, Viktoria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The use of the human neuroblastoma SH-SY5Y cell line for estimation of acute systemic toxicity in vitro.2007Konferansepaper (Fagfellevurdert)
    Abstract [en]

    Acute systemic toxicity, expressed as human lethal blood peak concentration or the dose inducing 50 % lethality in an animal population (LD50), can be estimated by general cytotoxicity tests using proliferating mammalian cell lines for 70-80 % of all chemicals. The cytotoxicity for the remaining chemicals over or under estimate the LD50 values/human lethal blood peak concentrations because of their very specific molecular targets or toxicokinetic features in vivo. The objective of the EU funded integrated project “ACuteTox” is to develop a strategy in which organ-specific endpoints and toxicokinetic features are taken into consideration in the in vitro prediction of acute systemic toxicity. The human neurotblastoma SH-SY5Y cell line was used as a model for studies on neurospecific targets, which are know to be crucial for survival. All endpoints were investigated after short exposure times (minutes to an hour) at concentrations of the test chemicals that did not affect the cell viability, measured as cell membrane leakage of lactate dehydrogenase. The effects of 23-26 compounds (drugs, pesticides and industrial chemicals) were studied on the cell membrane potential (CMP), voltage dependent Ca2+ channels (VDCC), muscarinic acetylcholine receptor (mAChR) function, acetylcholinesterase (AChE) activity and noradrenalin uptake. The results showed that the CMP was altered by atropine, amphetamine, mercury chloride, methadone, nicotine, pentachlorphenol, sodium lauryl sulphate (SLS) and verapamil, where as an effect on VDCC could be detected for amphetamine, atropine, colchicine, pentachlorphenol, SLS and verapamil. The mAChR function was measured as carbachol-induced Ca2+, i.e. activation of phospholipase C. Amphetamine, pentachlorphenol, SDS and verapamil attenuated the carbachol response by 50% at concentrations about 1 mM, but the specific mAChR antagonist atropine had the same effect at 3 nM. Nicotine, caffeine, pentachlorphenol, methadone, mercury chloride, SLS and the specific inhibitors physostigmine, dichlorvos and malathion attenuated the AChE activity at significantly non-cytotoxic concentrations in SH-SY5Y cells after 60 minutes of exposure. Parathion did not inhibit the AChE activity after 60 minutes exposure, but after 48 hr, indicating that oxidation of parathion to the active inhibitor paraoxon took place in the cell culture. This phenomenon was also observed for malathion, which displayed a lower EC50 value after the prolonged exposure time. The noradrenalin uptake was affected by atropine, caffeine, carbamazepine, amphetamine, diazepam, isopropanol, methadone, SLS and verapamil. A comparison of the active concentrations with the basal cytotoxicity measured as neutral red uptake in mouse fibroblast 3T3 cells indicated that the AChE assay is useful for detection of AChE inhibitors and possibly also AChR ligands. The VDCC endpoint was useful as an alert only for verapamil and the mAChR function was only specifically affected by atropine. The noradrenalin uptake indicated a clear alert for amphetamine and methadone, which was expected, but not for the other test compounds. These results indicate that the usefulness of these endpoints in a general test battery for estimation of acute systemic toxicity is limited, except for AChE activity measurements. However, the results clearly showed that the compounds with known mechanisms (e.g. atropine, verapamil, amphetamine and methodone ) displayed expected effects on their specific endpoints.

  • 168.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindegren, Heléne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Axelsson, Viktoria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity2010Inngår i: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 245, nr 2, s. 191-202Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The objective of the EU-funded integrated project ACuteTox is to develop a strategy in which general cytotoxicity, together with organ-specific toxicity and biokinetic features, are used for the estimation of human acute systemic toxicity. Our role in the project is to characterise the effect of reference chemicals with regard to neurotoxicity. We studied cell membrane potential (CMP), noradrenalin (NA) uptake, acetylcholine esterase (AChE) activity, acetylcholine receptor (AChR) signalling and voltage-operated calcium channel (VOCC) function in human neuroblastoma SH-SY5Y cells after exposure to 23 pharmaceuticals, pesticides or industrial chemicals. Neurotoxic alert chemicals were identified by comparing the obtained data with cytotoxicity data from the neutral red uptake assay in 3T3 mouse fibroblasts. Furthermore, neurotoxic concentrations were correlated with estimated human lethal blood concentrations (LC50). The CMP assay was the most sensitive assay, identifying eight chemicals as neurotoxic alerts and improving the LC50 correlation for nicotine, lindane, atropine and methadone. The NA uptake assay identified five neurotoxic alert chemicals and improved the LC50 correlation for atropine, diazepam, verapamil and methadone. The AChE, AChR and VOCC assays showed limited potential for detection of acute toxicity. The CMP assay was further evaluated by testing 36 additional reference chemicals. Five neurotoxic alert chemicals were generated and orphendrine and amitriptyline showed improved LC50 correlation. Due to the high sensitivity and the simplicity of the test protocol, the CMP assay constitutes a good candidate assay to be included in an in vitro test strategy for prediction of acute systemic toxicity.

  • 169.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Söderdahl, Therése
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jönsson, Gunn
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bratteng, Jan-Ove
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin-like growth factor type 1 prevents hyperglycemia-induced uncoupling protein 3 down-regulation and oxidative stress2004Inngår i: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 77, nr 2, s. 285-291Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Uncoupling proteins (UCPs) have been reported to decrease the mitochondrial production of reactive oxygen species (ROS) by lowering the mitochondrial inner membrane potential (MMP). We have previously shown that UCP3 expression is positively regulated by insulin-like growth factor-1 (IGF-1). The aim of this study was to investigate the role of UCPs in IGF-1-mediated protection from hyperglycemia-induced oxidative stress and neurodegeneration. Human neuroblastoma SH-SY5Y cells were differentiated with retinoic acid for 6 days, after which exposure to 8, 30, or 60 mM glucose with or without 10 nM IGF-1 was started. After 48-72 hr, the number of neurites per cell, UCP3 protein expression, MMP, and intracellular levels of ROS and total glutathione were examined. These studies showed that glucose concentration-dependently reduced the number of neurites per cell, with a 50% reduction at 60 mM. In parallel, the UCP3 protein expression was down-regulated, and the MMP was raised 3.5-fold, compared with those in cells incubated with 8 mM glucose. Also, the ROS levels were increased, showing a twofold maximum at 60 mM glucose. This was accompanied by a twofold elevation of total glutathione levels, confirming an altered cellular redox state. IGF-1 treatment prevented the glucose-induced neurite degeneration and UCP3 down-regulation. Furthermore, the MMP and the intracellular levels of ROS and glutathione were normalized to those of control cells. These data indicate that IGF-1 may protect from hyperglycemia-induced oxidative stress and neuronal injuries by regulating MMP, possibly by the involvement of UCP3.

  • 170.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tamm, Christoffer
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 32004Inngår i: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 88, nr 2, s. 462-468Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Uncoupling protein 3 (UCP3) is a mitochondrial protein with antioxidant properties and its regulation by factors promoting cell-survival may be important for protection of, for instance, neurons in states of oxidative stress. In the present study, we investigated regulatory pathways for UCP3 expression mediated by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1) in human neuroblastoma SH-SY5Y cells. Northern blot analysis and RT-PCR showed that treatment with 10 nm IGF-1 increased the UCP3 mRNA levels 2.5-fold after 5 h. Co-incubation with the phosphatidylinositol 3 (PI3)-kinase inhibitor LY294002 prohibited IGF-1-mediated induction of both UCP3 mRNA and protein in a concentration-dependent manner, with a complete blockage at 1 μm, as shown by RT-PCR and western blot analyses. The mitogen-activated protein (MAP) kinase kinase 1 (MKK1 or MEK) inhibitor PD98059 also decreased the UCP3 mRNA expression at 10 μm, however, this concentration only partly inhibited the protein expression. We conclude that IGF-1 enhanced UCP3 expression at transcriptional level, primarily through the PI3-kinase-dependent pathway and partly through the MAP kinase pathway.

  • 171.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tamm, Christoffer
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 3.2004Inngår i: J Neurochem, ISSN 0022-3042, Vol. 88, nr 2, s. 462-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Uncoupling protein 3 (UCP3) is a mitochondrial protein with antioxidant properties and its regulation by factors promoting cell-survival may be important for protection of, for instance, neurons in states of oxidative stress. In the present study, we investigated regulatory pathways for UCP3 expression mediated by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1) in human neuroblastoma SH-SY5Y cells. Northern blot analysis and RT-PCR showed that treatment with 10 nm IGF-1 increased the UCP3 mRNA levels 2.5-fold after 5 h. Co-incubation with the phosphatidylinositol 3 (PI3)-kinase inhibitor LY294002 prohibited IGF-1-mediated induction of both UCP3 mRNA and protein in a concentration-dependent manner, with a complete blockage at 1 microm, as shown by RT-PCR and western blot analyses. The mitogen-activated protein (MAP) kinase kinase 1 (MKK1 or MEK) inhibitor PD98059 also decreased the UCP3 mRNA expression at 10 microm, however, this concentration only partly inhibited the protein expression. We conclude that IGF-1 enhanced UCP3 expression at transcriptional level, primarily through the PI3-kinase-dependent pathway and partly through the MAP kinase pathway.

  • 172.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function: Reconnaissance for designing functional conjugates2008Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that one protein isoform is disease-related while another isoform is desirable. Splice switching opens the door to new drug targets, and antisense oligonucleotides (asONs), designed to switch splicing, are effective drug candidates. Cellular uptake of oligonucleotides(ONs) is poor, therefore utilization of cell-penetrating peptides (CPPs), well recognized for intracellular cargo delivery, is a promising approach to overcome this essential issue. Most CPPs are internalized by endocytosis, although the mechanisms involved remain controversial.

    Here, evaluation of CPP-mediated ON delivery over cellular membranes has been performed. A protocol that allows for convenient assessment of CPP-mediated cellular uptake and characterization of corresponding internalization routes is established. The protocol is based on both fluorometric uptake measurements and a functional splice-switching assay, which in itself is based on biological activity of conveyed ONs. Additionally, splice switching ONs (SSOs) have been optimized for high efficiency and specificity. Data suggest that SSO activity is improved for chimeric phosphorothioate SSOs containing locked nucleic acid (LNA) monomers. It is striking that the LNA monomers in such chimeric constructs give rise to low mismatch discrimination of target pre-mRNA, which highlight the necessity to optimize sequences to minimize risk for off-target effects.

    The results are important for up-coming work aimed at developing compounds consisting of peptides and novel synthetic nucleic acids, making these entities winning allies in the competition to develop therapeutics regulating protein expression patterns.

  • 173.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Atypical gene expression has a major influence on the disease profile of several severe human disorders. Oligonucleotide (ON) based therapeutics has opened an avenue for compensating deviant protein expression by acting on biologically important nucleic acids, mainly RNAs. Antisense ONs (asONs) can be designed to target complementary specific RNA sequences and thereby to influence the corresponding protein synthesis. However, cellular uptake of ONs is poor and is, together with the target specificity of the asONs, the major limiting factor for the development of ON based therapeutics.

    In this thesis, the mechanisms of well-characterized cell-penetrating peptides (CPPs) are evaluated and CPPs are adapted for cellular ON-delivery. The functionality of ON derivatives in cells is investigated and by optimization of asONs, targeting pre-messenger RNA, high efficiency and specificity is achieved. The optimization of the asONs is based on sequence design and through the choice of nucleic acid analogue composition. It is concluded that asONs, partly composed of locked nucleic acids are attractive for splice-switching applications but these mixmers must be designed with limited number of locked nucleic acid monomers to avoid risk for off-target activity. A protocol allowing for convenient characterization of internalization routes for CPPs is established and utilized. A mechanistic study on cellular CPP uptake and translocation of associated ON cargo reveals the importance of the optimal combination of for example charge and hydrophobicity of CPPs for efficient cellular uptake. Formation of non-covalent CPP:ON complexes and successful cellular delivery is achieved with a stearylated version of the well-recognized CPP, transportan 10.

    The results illustrate that CPPs and ON derivatives have the potential to become winning allies in the competition to develop therapeutics regulating specific protein expression patterns involved in the disease profile of severe human disorders.

  • 174.
    Guterstam, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Characterization of cellular internalization pathways for CPP-mediated oligonucleotide delivery2011Inngår i: Cell-penetrating peptides: Methods and Protocols / [ed] Ülo Langel, New York: Humana Press, 2011, s. 219-230Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    The methods for evaluating internalization pathways of cellular CPP-mediated ON delivery utilizing a pre-mRNA splice correction assay and fluorescence-based quantification are described. Examples for characterization of CPP uptake routes, employing various endocytosis inhibitors, and special treatment conditions are demonstrated. The methods are developed to characterize cellular delivery of pre-mRNA splice switching peptide nucleic acids conjugated to CPPs by disulfide bond.

  • 175.
    Guterstam, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindgren, Maria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tedebark, Ulf
    Wengel, Jesper
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers2008Inngår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 412, s. 307-313Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced beta-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2'-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.

  • 176.
    Guterstam, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Madani, Fatemeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hirose, Hisaaki
    Takeuchi, Toshihide
    Futaki, Shiroh
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate2009Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, nr 12, s. 2509-2517Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.

  • 177. Hamm, Jon
    et al.
    Sullivan, Kristie
    Clippinger, Amy J.
    Strickland, Judy
    Bell, Shannon
    Bhhatarai, Barun
    Blaauboer, Bas
    Casey, Warren
    Dorman, David
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swedish Toxicology Sciences Research Center (Swetox), Sweden.
    Garcia-Reyero, Natàlia
    Gehen, Sean
    Graepel, Rabea
    Hotchkiss, Jon
    Lowit, Anna
    Matheson, Joanna
    Reaves, Elissa
    Scarano, Louis
    Sprankle, Catherine
    Tunkel, Jay
    Wilson, Dan
    Xia, Menghang
    Zhu, Hao
    Allen, David
    Alternative approaches for identifying acute systemic toxicity: Moving from research to regulatory testing2017Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 41, s. 245-259Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Acute systemic toxicity testing provides the basis for hazard labeling and risk management of chemicals. A number of international efforts have been directed at identifying non-animal alternatives for in vivo acute systemic toxicity tests. A September 2015 workshop, Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing, reviewed the state-of-the-science of non-animal alternatives for this testing and explored ways to facilitate implementation of alternatives. Workshop attendees included representatives from international regulatory agencies, academia, nongovernmental organizations, and industry. Resources identified as necessary for meaningful progress in implementing alternatives included compiling and making available high-quality reference data, training on use and interpretation of in vitro and in silico approaches, and global harmonization of testing requirements. Attendees particularly noted the need to characterize variability in reference data to evaluate new approaches. They also noted the importance of understanding the mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Workshop breakout groups explored different approaches to reducing or replacing animal use for acute toxicity testing, with each group crafting a roadmap and strategy to accomplish near-term progress. The workshop steering committee has organized efforts to implement the recommendations of the workshop participants.

  • 178.
    Hankel, Sabine
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Studies on inflammatory myopathies and adult neurogenesis2006Licentiatavhandling, monografi (Annet vitenskapelig)
  • 179.
    Hansen, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Internalization mechanisms of cell-penetrating peptides: Structure-activity relationships, cellular distribution and membrane toxicity studies2008Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Transport of substances with pharmaceutical potential over the plasma membrane has been pushed a great step forward with the discovery of short cationic polypeptides, known as cell-penetrating peptides (CPP). Since then, the internalization mechanism and cargo delivery properties of CPPs has been extensively studied. Unfortunately, no consensus over the internalization mechanism has been achieved so far. Much attention has been paid to the delivery of various macromolecules into the cytoplasm and other subcellular compartments with the help of CPPs, while the structural requirements and toxicity of the peptides and their conjugates has not been studied as thoroughly.

    The focus of this thesis is on the characterization of internalization mechanism, toxicity and prediction of CPPs as currently most discussed issues in the field of research.

    The studies included in the present thesis indicate that for the majority of CPPs the uptake is dependent of endocytic processes. However, these studies do not reveal one major endocytosis pathway, responsible for the uptake of all tested CPPs. Instead it is most likely that different mechanisms are involved in the uptake of different CPPs. Peptides with apparent amphipathic natures are mainly internalized via clathrin-mediated endocytosis while the CPPs with low amphipathic moment utilize macropinocytosis.

    Disturbance of plasma membrane by CPPs in different cell lines was also studied. Results show that peptides with higher amphipathic moment have, in general, higher toxicity, both long- and short-term. However, the amphipathic nature of CPPs is not the only criterion for membrane perturbation. Results also demonstrated the difference in membrane perturbation in different cell lines.

    Taken together the studies involved in this thesis provide useful information about the internalization mechanisms and cytotoxicity of cell-penetrating peptides.

  • 180.
    Hansen, Mats
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Eriste, Elo
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    The internalization mechanisms and bioactivity of the cell-penetrating peptides2009Inngår i: Peptides as drugs: Discovery and Development / [ed] Bernd Groner, Weinheim: Wiley-VCH Verlagsgesellschaft, 2009, s. 125-143Kapittel i bok, del av antologi (Fagfellevurdert)
  • 181.
    Hansen, Mats
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, H.
    Guterstam, P.
    Holm, T.
    Langel, Ü
    EL Andaloussi, S.
    Distinct uptake routes of CPPs and differential dependence of heparan sulfate for internalizationManuskript (Annet vitenskapelig)
  • 182.
    Hansen, Mats
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kilk, Kalle
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Predicting cell-penetrating peptides2008Inngår i: Advanced Drug Delivery Reviews, ISSN 0169-409X, E-ISSN 1872-8294, Vol. 60, nr 4-5, s. 572-579Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Possibility to predict short peptide sequences capable to penetrate the plasma membrane opens new opportunities for developing peptide based intracellular delivery vectors, called cell-penetrating peptides (CPPs). Predictions of CPPs, however are often based on trial and error and may not always lead to new potent sequences. In this review we discuss different problems associated with CPP prediction. Additionally, the used methods of CPP prediction are compared. Also, a few suggestions are made for designing new CPP sequences and improvement of predictions.

  • 183. Hassane, Fatouma Said
    et al.
    Abes, Rachida
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lehto, Taavi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sillard, Rannar
    Langel, Ulo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lebleu, Bernard
    Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides2011Inngår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 153, nr 2, s. 163-172Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Conjugates of cell-penetrating peptides (CPP) and splice redirecting oligonucleotides (ON) display clinical potential as attested by in vivo experimentation in murine models of Duchenne muscular dystrophy. However, micromolar concentrations of these conjugates are required to obtain biologically relevant responses as a consequence of extensive endosomal sequestration following endocytosis. Recent work from our group has demonstrated that appending stearic acid to CPPs increases their efficiency and that the inclusion of pH titrable entities leads to further improvement. Moreover, these modified CPPs form non covalent complexes with charged ON analogs or siRNAs, which allows decreasing the concentrations of ONs by nearly one log. These modified CPPs and the parent peptides have been compared here in the same in vitro model in terms of cell uptake, trafficking and splicing redirection activity. The increased splicing redirection activity of our modified CPPs cannot be explained by differences in cell uptake but rather by their enhanced ability to escape from endocytic vesicles. Accordingly, a clear correlation between membrane destabilizing activity and splicing redirection was observed using a liposome leakage assay. Studies of cellular trafficking for the most active PF6:ON complexes indicate uptake by clathrin-mediated endocytosis using either FACS cell uptake or a splicing redirection functional assay. Acidification of intracellular vesicles and membrane potential were found important for splicing redirection but not for cell uptake. These results do confirm that the increased potency of PF6:ON complexes is not due to the use of a non endocytic route of cell internalization as proposed for some CPPs.

  • 184. Hawes, Jessica
    et al.
    Brunzell, Darlene
    Narasimhaiah, Roopashree
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Wynick, David
    Picciotto, Marina
    Galanin Protects Against Behavioral and Neurochemical Correlates of Opiate Reward2008Inngår i: Neuropsychopharmacology, ISSN 0893-133X, E-ISSN 1740-634X, Vol. 33, nr 8, s. 1864-1873Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The mechanisms underlying responses to drugs of abuse have been widely investigated; however, less is known about pathways normally protective against the development of drug reinforcement. These pathways are also important since they may regulate individual differences in vulnerability to addiction. The neuropeptide galanin and its binding sites are expressed in brain areas important for drug reward. Previous studies have shown that centrally infused galanin attenuates morphine place preference and peripheral injection of galnon, a galanin agonist, decreases opiate withdrawal signs. The current studies in galanin knockout (GKO) mice examined the hypothesis that galanin is an endogenous negative regulator of opiate reward and identified downstream signaling pathways regulated by galanin. We show that GKO mice demonstrate increased locomotor activation following morphine administration, which is inhibited by acute administration of galnon. GKO mice also show enhanced morphine place preference, supporting the idea that galanin normally antagonizes opiate reward. In addition, morphine-induced ERK1/2 phosphorylation was increased in the VTA of both wild-type and GKO mice, but only the GKO mice showed increases in ERK1/2 and CREB phosphorylation in the amygdala or nucleus accumbens. Furthermore, a single systemic injection of galnon in GKO mice was sufficient to reverse some of the biochemical changes brought about by morphine administration. These data suggest that galanin normally attenuates behavioral and neurochemical effects of opiates; thus, galanin agonists may represent a new class of therapeutic targets for opiate addiction.

  • 185.
    Hegazy, Usama M.
    et al.
    National Research Centre (NRC), Egypt.
    Musdal, Yaman
    Hacettepe University, Turkey.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hidden Allostery in Human Glutathione Transferase P1-1 Unveiled by Unnatural Amino Acid Substitutions and Inhibition Studies2013Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 9, s. 1509-1514Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kat and 300-fold increased K-M(GSH) values, displays an apparent Hill coefficient of 0.82 +/- 0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency k(cat)/K-M(GSH) to near the wild-type value but still yields Hill coefficients of 0.61 +/- 0.08 and 0.86 +/- 0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99 +/- 0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.

  • 186.
    Helmfors, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides: an uptake mechanism & a new endosomolytic peptide2013Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Peptide-based drugs have slowly begun migrating from laboratories into pharmacies and now there are several on the market. However, currently only one gene based therapy that is relies on a viral delivery vector has been approved. The long-term goal of our research is to leverage the cell-penetrating peptide (CPP) technology into a potent, safe and simple delivery vector for oligonucleotide (ON) based therapies.

    Cell-penetrating peptides have been actively researched for more than 20 years, and many CPPs have been discovered. However, it is not fully understood how the peptides are able to enter cells. In this thesis we present a novel receptor for CPP:ON complexes. Pharmacological inhibition and siRNA knockdown of the class A scavenger receptors (SCARAs) demonstrate that these receptors are the main pathway by which CPP:ON complexes are taken up. As the intracellular fate of particles taken up by (receptor mediated) endocytosis is entrapment in endosomes this thesis also presents a new peptide for ON delivery that has endosomolytic properties. Additionally this new peptide (PepFect 15) is also taken up via receptor-mediated endocytosis by the SCARAs. 

  • 187.
    Helmfors, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides: Uptake mechanism and the role of receptors2015Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Genes are the major regulators of biological processes in every living thing. Problems with gene regulation can cause serious problems for the organism; for example, most cancers have some kind of genetic component. Regulation of biological processes using oligonucleotides can potentially be a therapy for any ailment, not just cancer. The problem so far has been that the targets for oligonucleotide-based therapies all reside on the inside of cells, because the cellular plasma membrane is normally impermeable to large and charged molecules (such as oligonucleotides) a delivery method is needed. Cell-penetrating peptides are a class of carrier molecules that are able to induce the cellular membrane into taking them and their cargo molecules into the cells. Understanding how and why cell-penetrating peptides work is one of the first and most important steps towards improving them to the point where they become useful as carriers for oligonucleotide-based therapies. This thesis is comprised of four scientific papers that are steps toward finding an uptake mechanism for cell-penetrating peptides that have been non-covalently complexed with oligonucleotides. In Paper I, we show that the scavenger receptors are responsible for uptake of the cell-penetrating peptide PepFect14 in complex with a short single-stranded oligonucleotide. Paper II expands upon this first finding and shows that the same receptors are key players in the uptake of several other cell-penetrating peptides that have been complexed with either, long double-stranded plasmid DNA or short double-stranded RNA. Paper III improves the luciferase-based assay for short oligonucleotide delivery by increasing the throughput 4-fold and reducing the cost by 95 %. The fourth manuscript uses the assay developed in paper III to investigate the effects on cell-penetrating peptide-mediated delivery by each of the constituents of a 264-member library of ligands for G-protein coupled receptors. We identify three ligands that dose-dependently increase the luciferase expression compared to control cells. These three ligands are one positive-, one negative allosteric modulator of metabotropic glutamate receptor 5 and one antagonist of histamine receptor 3.

  • 188.
    Helmfors, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eriksson, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides2015Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 484, s. 136-142Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z' scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.

  • 189.
    Helmfors, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    GPCR-ligands influence the short oligonucleotide transfection efficacy of the cell-penetrating peptide; Pepfect14Manuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. We have previously shown that inhibition or knock-down of scavenger receptor type A results in a decreased oligonucleotide uptake. The remaining question is if the scavenger receptors are the only cell-surface receptors that can affect the uptake. By utilizing an optimized, higher throughput assay, for short oligonucleotide delivery using cell-penetrating peptides, and simultaneously adding a G-protein coupled receptor-ligand library. We show that two allosteric modulators (MPEP and VU 0357121) of metabotropic glutamate receptor type 5 and one histamine H3 receptor antagonist (Ciproxifan) have effects that can increase the transfection efficacy of PepFect in complex with a short single stranded oligonucleotide. Five different estrogen receptor ligands have negative effects on the transfection efficacy.

  • 190.
    Helmfors, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Recent developments in applications of cell penetrating peptides uptake mechanisms and oligonucleotide delivery2012Inngår i: Chimica oggi, ISSN 0392-839X, E-ISSN 1973-8250, Vol. 30, nr 2, s. 10-12Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Here we report on recent developments in cell-penetrating peptide mediated delivery of siRNA and other oligonucleotides. We also report on the latest discoveries regarding the debated uptake mechanism of cell-penetrating peptides. For the first time evidence of a cell surface receptor involvement in the uptake of cell-penetrating peptide/oligonucleotide complexes has been described, indicating that properties of the cargo are likely crucial for which pathway is responsible for uptake.

  • 191.
    Helmfors, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Tartu University, Estonia.
    SCARA Involvement in the Uptake of Nanoparticles Formed by Cell-Penetrating Peptides2015Inngår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, Vol. 1324, s. 163-174Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    The investigation of uptake mechanisms for cell-penetrating peptides (CPPs) is and has been an ongoing project for as long as the peptides have been known, a time period that now spans over two decades. The ultimate answer is yet to be revealed and the current understanding is that no "one" mechanism will ever be found. The reason for this is that the uptake mechanism seems to be dependent on a multitude of factors that include which CPP, what cells are used, whether or not there is cargo and what the cargo is. CPPs are capable of delivering a variety of bio-macromolecules that are by themselves unable to enter into cells. Our group has reported on many different peptides in recent years, many aimed at delivering various oligonucleotide-based cargoes. These peptides have utilized the inherent positive charge of the peptides and some rationally designed modifications to non-covalently complex oligonucleotides and bring them into cells. In this chapter, we present a brief overview of the current proposals for the uptake mechanisms of CPPs and describe methods for detecting and evaluating the role of scavenger receptor class A receptors in the uptake of non-covalent cell-penetrating peptide:oligonucleotide complexes.

  • 192.
    Heléne, Lindegren
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lilja, Johanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    A NOVEL MECHANISTIC PATHWAY FOR SURFACTANT-INDUCED NOCICEPTION2007Inngår i: 25th SSCT annual workshop, 2007Konferansepaper (Annet (populærvitenskap, debatt, mm))
    Abstract [en]

    The pain receptor transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca2+ influx with secondary effects leading to neurogenic inflammation. Here we report specific activation of TRPV1 by detergent-containing hygiene products measured as intracellular Ca2+ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. Commercial hygiene products were applied to the cells in diluted solutions and stimulation was monitored during exposure as an increase in the intracellular free Ca2+ levels by using the Fura 2 fluorescence assay and semi-HTS fluorometer for 96 well plates. Co-application with the TRPV1 antagonist capsazepine revealed if the response was specific for the receptor. The response was quantified as the product induced Ca2+ influx during 2 minutes in relation to the maximum response induced by the TRPV1 agonist capsaicin. The results show that shampoos and soaps induced a TRPV1 mediated Ca2+ influx whereas children products marketed as “painless” (containing lower concentration of detergents), and conditioners (without detergents) did not induce specific TRPV1 activation. Furthermore, low concentrations of the detergent sodium lauryl sulfate (SLS) dose-dependently induced Ca2+ influx that could be addressed to TRPV1. These results reveal a novel mechanistic pathway for surfactant-induced nociception, which may be an important endpoint in in vitro test batteries, as alternatives to Draize’s rabbit eye test, for classification of mild eye irritating products.

  • 193.
    Holback, Sofia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Proteolytic processing of the Alzheimer APP protein family during neuronal differentiation2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Increased amyloid-β (Aβ) load in the brain, neurite degeneration, neuronal loss, and decreased levels of several neurotrophins are among the characteristics of Alzheimer’s disease (AD). Generation of Aβ occurs when the amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases in the amyloidogenic pathway. However, Aβ formation is prevented if APP is cleaved by α- and γ- secretases in the non-amyloidogenic pathway. The normal function of APP is still not fully known. It seems clear that the different fragments that are produced during proteolytic processing have different bioactive properties. APP and its metabolites have been implicated in neurite outgrowth, synaptogenesis, cell adhesion, neuroprotection and apoptosis.

    The aim of this thesis was to investigate how neurotrophic factors affect the synthesis and processing of APP and its two mammalian paralogues the APP-like protein-1 and-2 (APLP1 and APLP2). We also wanted to determine how the expression levels of α- and β- secretases were affected in response to these factors. In addition, we wanted to analyze if the levels and function of the most well characterized APP adaptor protein, Fe65, was regulated during neuronal differentiation.

    Our results show that retinoic acid (RA), insulin-like growth factor-1 (IGF-1), and brain derived neurotrophic factor (BDNF) all regulate expression levels and processing of the APP protein family. Interestingly, the increased processing of the APP family involves different signaling pathways. The PI3-K/Akt pathway is involved in IGF-1-induced APP and APLP1, but not APLP2, processing. In addition, RA-induced expression of the α-secretase, a disintegrin and metalloproteinase (ADAM) 10 is dependent on PI3-K, whereas PKC is involved in RA-induced expression of another α-secretase, ADAM17/TACE. Furthermore, we present evidence that maturation of the adaptor protein Fe65, as well as its docking to APP, increases concomitant with neuronal differentiation.

  • 194.
    Holback, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Adlerz, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gatsinzi, Tom
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The APP processing enzyme ADAM10 is up-regulated by retinoic acid in a PI3-kinase-dependent mannerInngår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468Artikkel i tidsskrift (Fagfellevurdert)
  • 195.
    Holback, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Adlerz, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gatsinzi, Tom
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jacobsen, Kristin T.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid2008Inngår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 365, nr 2, s. 298-303Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 196.
    Holback, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Adlerz, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Increased processing of APLP2 and APP with concomitantformation of APP intracellular domains in BDNF and retinoic acid-differentiated human neuroblastoma cells2005Inngår i: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 95, nr 4, s. 1059-1068Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 197.
    Holback, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Koistinen, Niina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jacobsen, Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Retinoic acid stimulates maturation of the adaptor protein Fe65 and its binding to the amyloid precursor proteinManuskript (preprint) (Annet vitenskapelig)
    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.

  • 198.
    Holm, Tina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides: Uptake, stability and biological activity2011Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have emerged as a group of remarkable delivery vectors for various hydrophilic macromolecules, otherwise excluded from cells due to the protective plasma membrane. Unbiased conclusions regarding e.g. uptake mechanism, intracellular distribution and cargo delivery efficacy is complicated by the use of different methodological parameters by different laboratories. The first paper in this thesis introduced unifying protocols enabling comparison of results from different research groups. One of these methods, HPLC, was used in paper II to investigate CPP uptake and degradation in yeasts. Both parameters varied depending on peptide and yeast species; however pVEC emerged as a promising delivery vector in yeast since it internalized into both species tested without concomitant degradation. Protein mimicry was another investigated phenomenon and in paper III a 22-mer peptide from the p14Arf protein (Arf (1-22)) was found to be sufficient for retaining its function as a tumor suppressor. This peptide comprised a combination of apoptogenic property and CPP in one unity, thus providing opportunity to conjugate cytotoxic agents boosting the tumoricidal activity. Surprisingly, a partially inverted control peptide to Arf (1-22), called M918, was found to be an extraordinary CPP. In paper IV, it was shown to be superior to well-established CPPs in delivery of both peptide nucleic acids and proteins. Albeit the promising results these two peptides displayed, their utility in vivo, as with all peptides, is hampered by rapid degradation. With the aim of improving their stability, Arf (1-22) and M918 were synthesized with D-amino acids in the reverse order, a modification called retro-inverso (RI) isomerization. Their cell-penetrating ability was retained, but the treated cells displayed unexpected morphological alterations indicative of apoptosis. The presented results demonstrate the versatility of CPPs, functioning as vectors in both yeast and mammalian cells and as protein mimicking peptides with biological activity. Their potential as drug delivery agents is obvious; however, peptide degradation is an issue that requires further improvements before clinical success is in reach.

  • 199.
    Holm, Tina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bruchmann, Julia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Scheynius, Annika
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides as antifungals towards Malassezia sympodialis2012Inngår i: Letters in Applied Microbiology, ISSN 0266-8254, E-ISSN 1472-765X, Vol. 54, nr 1, s. 39-44Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aim: To determine whether different antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) are able to inhibit the growth of the commensal yeast Malassezia sympodialis, which can act as a trigger factor in different skin disorders, such as atopic eczema (AE), seborrhoeic eczema (SE) and dandruff. Methods and results: The antifungal activity of 21 different AMPs and CPPs was investigated by microdilution assay and plate counting to determine the number of colony forming units. Five CPPs and one AMP showed fungicidal activity at submicromolar concentrations. Importantly, no membrane damage on human keratinocytes was detected after peptide treatment. Conclusions: Several CPPs, while being nontoxic to mammalian cells, possess growth inhibitory activity on the very stringent yeast M. sympodialis. Significance and impact of study: Our findings that the CPPs and one AMP that are harmless towards mammalian cells act as antifungal agents against sympodialis opens up the possibility to use these in the treatment for AL, SE and dandruff. To our knowledge, this is the first time peptides have been identilied as antilungal agents against sympodialis. Further studies to ekicidate the mechanism are warranted.

  • 200.
    Holm, Tina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Comparison of CPP uptake methods2011Inngår i: Cell-penetrating peptides: Methods and Protocols, New York: Humana Press, 2011, s. 207-217Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    In the last 15 years, an ever expanding pool of cell-penetrating peptides (CPPs) has been discovered and recently focus has shifted towards improving already existing CPPs by different modifications. Since the number of published peptide sequences with cell-penetrating ability is now reaching several hundreds, the consensus methods to compare the efficacy of these is clearly needed. Many research groups are evaluating the applicability of CPPs as drug delivery vectors, all having their preferred methods of assessing uptake and intracellular distribution. Even when applying the same method, the use of different cell lines, peptide concentrations, exposure conditions, etc. are complicating comparison of data between different groups. This book is a welcome contribution to the CPP research field, hopefully paving the way for standardized protocols to be used in the future. Some of the most common methods used to this date are presented and compared in this chapter.

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