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  • 251. Karelson, Ello
    et al.
    Fernaeus, Sandra
    Reis, Katarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bogdanovic, Nenad
    Land, Tiit
    Stimulation of G-proteins in human control and Alzheimer's disease brain by FAD mutants of APP(714-723): implication of oxidative mechanisms2005Inngår i: Journal of Neuroscience Research, ISSN 0360-4012, Vol. 79, nr 3, s. 368-374Artikkel i tidsskrift (Fagfellevurdert)
  • 252. Keimpema, Erik
    et al.
    Zheng, Kang
    Barde, Swapnali Shantaram
    Berghuis, Paul
    Dobszay, Marton B.
    Schnell, Robert
    Mulder, Jan
    Luiten, Paul G. M.
    Xu, Zhiqing David
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lu, Bai
    Hokfelt, Tomas
    Harkany, Tibor
    GABAergic Terminals Are a Source of Galanin to Modulate Cholinergic Neuron Development in the Neonatal Forebrain2014Inngår i: Cerebral Cortex, ISSN 1047-3211, E-ISSN 1460-2199, Vol. 24, nr 12, s. 3277-3288Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The distribution and (patho-) physiological role of neuropeptides in the adult and aging brain have been extensively studied. Galanin is an inhibitory neuropeptide that can coexist with.-aminobutyric acid (GABA) in the adult forebrain. However, galanin's expression sites, mode of signaling, impact on neuronal morphology, and colocalization with amino acid neurotransmitters during brain development are less well understood. Here, we show that galaninergic innervation of cholinergic projection neurons, which preferentially express galanin receptor 2 (GalR2) in the neonatal mouse basal forebrain, develops by birth. Nerve growth factor (NGF), known to modulate cholinergic morphogenesis, increases GalR2 expression. GalR2 antagonism (M871) in neonates reduces the in vivo expression and axonal targeting of the vesicular acetylcholine transporter (VAChT), indispensable for cholinergic neurotransmission. During cholinergic neuritogenesis in vitro, GalR2 can recruit Rho-family GTPases to induce the extension of a VAChT-containing primary neurite, the prospective axon. In doing so, GalR2 signaling dose-dependently modulates directional filopodial growth and antagonizes NGF-induced growth cone differentiation. Galanin accumulates in GABA-containing nerve terminals in the neonatal basal forebrain, suggesting its contribution to activity-driven cholinergic development during the perinatal period. Overall, our data define the cellular specificity and molecular complexity of galanin action in the developing basal forebrain.

  • 253.
    Kilk, Kalle
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides and bioactive cargoes: Strategies and mechanisms2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The cell membrane is an impermeable barrier for most macromolecules. Recently discovered cell-penetrating peptides (CPPs) have gained lot of attention because they can cross the membrane, and even more, carry cargoes with them. How CPPs enter cells is still not clear, while the delivery of different cargoes has been convincingly shown. This thesis concentrates on evaluating CPPs as vectors for different biologically relevant cargoes. Proposed internalisation mechanisms are reviewed as well as cargo coupling strategies. Biological activities of antisense oligonucleotides delivered by CPPs have been of particular interest and are explained in greater details.

    A new CPP, pIsl, was derived from Islet-1 transcription factor, and compared to archetypical CPPs like penetratin and transportan. All three peptides resided in the headgroup region of lipid bilayers in model membranes. However, penetratin and pIsl did only interact with negatively charged membranes, while transportan did not distinguish negatively charged and neutral membranes. This suggests different translocation pathways for different CPPs. Biotinylated pIsl and penetratin were complexed with avidin, and uptake of avidin into the human melanoma cell line Bowes was observed in both cases. This means that the protein is not unfolded during the translocation process, which is important in delivery of other, biologically active proteins.

    Transportan and its analogue TP10 were used for peptide nucleic acid (PNA) antisense oligonucleotide delivery. First, eight human galanin receptor type 1 targeting PNA oligomers were designed, conjugated to transportan and assayed for antisense efficiency. In contrary to avidin-biotinylated peptide conjugate, a covalent bond between PNA oligomers and the transport peptide was necessary for cellular uptake of oligomers. A common problem in antisense technology is inactivity of antisense oligonucleotides due to the secondary structure of the target. Efficiencies of tested galanin receptor type 1 targeting PNA oligomers varied over two orders of magnitude. The most efficient oligomers were targeting coding sequence regions 24-38 and 27-38, and had EC50 values 70 and 80 nM, respectively.

    TP10-antisense PNA oligomer conjugates were targeted also to L-type voltage dependent Ca2+ channel subunits CaV1.2 and CaV1.3. Specific down-regulation of respective proteins was demonstrated by immunohistochemistry. Physiological response to the down-regulation of either of Ca2+ channels was studied by alteration of flexor reflex sensitisation. Rats treated with either of the antisense PNA, but not with scrambled PNA lost the action potential windup phenomenon. In conjunction with a variety of drugs, modulating the conductivity and excitability of neuronal membranes, a central role of L-type CaV channels in sensitisation was confirmed. Nevertheless, also N-methyl-D-aspartate and glycine receptors were found to be required.

    Finally, delivery of plasmids by TP10 was evaluated. In contrary to many similar CPPs, TP10 was incapable to translocate plasmids to cells. However, addition of TP10 or a TP10-PNA conjugate to polyethyleneimine-condensed plasmids increased the expression of reporter genes.

    In summary, different types of cargoes have been delivered by CPPs and different cargo coupling strategies have been used. CPP-mediated antisense oligonucleotide delivery has been used to identify accessible sites in human galanin receptor type 1 mRNA and to determine the role of L-type voltage dependent Ca2+ channels in axon potential windup.

  • 254. Kilk, Kalle
    et al.
    Mahlapuu, Riina
    Soomets, Ursel
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Analysis of in vitro toxicity of five cell-penetrating peptides by metabolic profiling2009Inngår i: Toxicology, ISSN 0300-483X, E-ISSN 1879-3185, Vol. 265, nr 3, s. 87-95Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are promising candidates for safe and efficient delivery vectors for a wide range of cargoes. However, any compound that is internalized into cells may affect the cell homeostasis. The plethora of possible biological responses makes large scale “omics” studies appealing approaches for hunting any unsuspected side-effects and evaluate the toxicity of drug candidates. Here we have compared the alterations in cytosolic metabolome of CHO cells caused by five representatives of the most common CPPs: transportan (TP), penetratin (pAntp), HIV Tat derived peptide (pTat), nonaarginine (R9) and model amphipathic peptide (MAP). Analysis was done by liquid chromatography–mass spectrometry techniques, principal component analysis and heatmap displays. Results showed that the intracellular metabolome was the most affected by TP followed by pTat and MAP. Only minor changes could be associated with pAntp or R9 treatment. The cells could recover from a treatment with 5 μM TP, but no recovery was observed at higher concentration. Both metabolomic and control experiments showed that TP affected cellular redox potential, depleted energy and the pools of purines and pyrimidines. In conclusion, we have performed a metabolomic analysis comparing the safety of cell-penetrating peptides and demonstrate the toxicity of one of them.

  • 255. Kim, Tae Kyung
    et al.
    Sul, Jai-Yoon
    Helmfors, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kim, Junhyong
    Eberwine, James
    Dendritic Glutamate Receptor mRNAs Show Contingent Local Hotspot-Dependent Translational Dynamics2013Inngår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 5, nr 1, s. 114-125Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Protein synthesis in neuronal dendrites underlies long-term memory formation in the brain. Local translation of reporter mRNAs has demonstrated translation in dendrites at focal points called translational hotspots. Various reports have shown that hundreds to thousands of mRNAs are localized to dendrites, yet the dynamics of translation of multiple dendritic mRNAs has remained elusive. Here, we show that the protein translational activities of two dendritically localized mRNAs are spatiotemporally complex but constrained by the translational hotspots in which they are colocalized. Cotransfection of glutamate receptor 2 (GluR2) and GluR4 mRNAs (engineered to encode different fluorescent proteins) into rat hippocampal neurons demonstrates a heterogeneous distribution of translational hotspots for the two mRNAs along dendrites. Stimulation with s-3,5-dihydroxy-phenylglycine modifies the translational dynamics of both of these RNAs in a complex saturable manner. These results suggest that the translational hotspot is a primary structural regulator of the simultaneous yet differential translation of multiple mRNAs in the neuronal dendrite.

  • 256. Kinsner-Ovaskainen, Agnieszka
    et al.
    Clemedson, Cecilia
    Cole, Thomas
    Clothier, Richard
    Coecke, Sandra
    O’Connor, José-Enrique
    Blaauboer, Bas
    Gómez-Lechón, Maria-José
    Vericat, Joan-Albert
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ryan, Michael
    Castell, José
    Risteli, Leila
    Wendel, Albrecht
    Optimisation and pre-validation of an in vitro test strategy for predicting human acute toxicity: Progress of the “A-Cute-Tox” project2007Konferansepaper (Annet vitenskapelig)
  • 257. Kjellander, Marcus
    et al.
    Mazari, Aslam M. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Boman, Mats
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Johansson, Gunnar
    Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability2014Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, s. 59-63Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via c-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.

  • 258.
    Kmiec, Beata
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Teixeira, Pedro F.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berntsson, Ronnie P. -A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Murcha, Monika W.
    Branca, Rui M. M.
    Radomiljac, Jordan D.
    Regberg, Jakob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Svensson, Linda M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bakali, Amin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lehtio, Janne
    Whelan, James
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts2013Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, nr 40, s. E3761-E3769Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 angstrom, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 angstrom(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.

  • 259.
    Koistinen, Niina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The adaptor protein Fe65 and APP processing2014Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The amyloid precursor protein (APP) protein has been in the limelight of research on Alzheimer´s disease (AD) pathogenesis because its proteolytic processing gives rise to the neurotoxic amyloid β (Aβ) peptide, the main constituent of amyloid plaques in the brains of AD patients. APP is sequentially processed by at least three different proteases termed α-, β-, and γ-secretases. The proteolytic processing of APP can be divided into two different pathways, the non-amyloidogenic and the amyloidogenic. Whether APP is processed by the non-amyloidogenic or the amyloidogenic pathway is highly dependent on colocalization of APP with the different processing enzymes. Hence, understanding the mechanism underlying regulation of APP trafficking and its related secretases is of great importance in our understanding of AD and AD pathogenesis. The aim of this thesis was to study the processing and trafficking of APP, how it may be regulated by the interaction with the adaptor protein, Fe65, and by a novel type of posttranslational modification, O-GlcNAcylation. We have used the human neuroblastoma cell line SH-SY5Y as a modell system to investigate the effect of Fe65 knock-down on APP processing. Our results showed that Fe65 knockdown did not have any effect on sAPPα secretion. However, decreased levels of C83 and C99 were observed, suggesting that Fe65 has a stabilizing effect on the C-terminal fragments. Furthermore, we investigated the effects of RA-induced neronal differentiation on Fe65 expression. We observed increased protein levels of Fe65 and an electrophoretic mobility shift due to increased phosphorylation of Fe65. O-GlcNAcylation is a dynamic posttranslational modification regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the effect of O-GlcNAcylation on APP trafficking and processing, SH-SY5Y cells were treated with PUGNAc, an OGA inhibitor, to increase the cellular levels of O-GlcNAc. The results revealed that cell surface localization of mature APP was significantly enhanced without any affect on the total levels of APP. We further show evidence that ADAM10 is O-GlcNAcylated and that the effect of O-GlcNAcylation on APP processing is neuron-specific.

  • 260.
    Koistinen, Niina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The amyloid-β precursor protein (APP)-binding protein Fe65 and APP processing2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of neurotoxic amyloid-β (Aβ) peptide. Aβ is generated by sequential cleavage of the amyloid-β precursor protein (APP) by β- and then γ-secretase. However, APP can also be processed by α- and γ-secretase, instead resulting in generation of neuroprotective sAPPα. Increased APP phosphorylation and altered expression levels of the brain enriched Fe65 protein have been observed in the brains of AD patients. Fe65 can not only interact with membrane tethered APP, but can also localized into the nucleus and act as a transcriptional regulator together with the APP intracellular domain (AICD), generated after γ-secretase processing. How APP processing, APP/Fe65 interaction, and the nuclear AICD/Fe65 complex is regulated has not yet been fully understood. The aim of this thesis was therefore to further elucidate how Fe65 is regulated and how APP Ser675 phosphorylation affects APP processing.

    We could identify several factors regulating Fe65. First, we identified that neuronal differentiation induces Fe65 phosphorylation (paper I), and that phosphorylated forms of Fe65 were preferentially localized outside the nucleus (paper II). Second, we found that the APP binding PTB2 domain of Fe65, rather than the previously proposed N-terminal WW domain, is important for the nuclear localization of Fe65 (paper II). In addition, we surprisingly found that mutation of S228 in the Fe65 N-terminus could increase the APP/Fe65 interaction (paper III). Third, both α- and γ-secretase inhibitors decreased Fe65 nuclear localization similarly, indicating an important role of α-secretase in regulating Fe65 nuclear localization (papers II and III). Lastly, we could in paper IV for the first time show that phosphorylation of APP at Ser675 regulates APP processing at the plasma membrane, resulting in reduced levels of sAPPα. These results, together with the observation that APP Ser675 phosphorylation occur in AD brains, suggest that Ser675 phosphorylation could contribute to AD pathology by decreasing α-secretase processing and instead increasing the levels of Aβ.

    In summary these studies have contributed to understanding of APP processing and the interplay between Fe65 and APP, two suggested key players in AD. 

  • 261.
    Koistinen, Niina A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bacanu, Smaranda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Phosphorylation of Fe65 amyloid precursor protein-binding protein in response to neuronal differentiation2016Inngår i: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 613, s. 54-59Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 262.
    Koistinen, Niina A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Edlund, Anna K.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Menon, Preeti K.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ivanova, Elena V.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bacanu, Smaranda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Nuclear localization of amyloid-beta precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis2017Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, nr 3, artikkel-id e0173888Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 263.
    Koistinen, Niina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holback, Sofia
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    siRNA knock-down of Fe65 in SH-SY5Y cells decreases the levels of C-terminal fragments of APP without any effect on sAPPα secretionManuskript (preprint) (Annet vitenskapelig)
    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.

  • 264.
    Koistinen, Niina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Menon, Preeti
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ivanova, Elena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kumcu, Michael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ström, Anna-Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    ADAM10 dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 is attenuated in neuronally differentiated SH-SY5Y cellsManuskript (preprint) (Annet vitenskapelig)
    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.      

  • 265.
    Koistinen, Niina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Menon, Preeti
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ström, Anna-Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    APP Ser675 phosphorylation affects α-secretase processing resulting in decreased secretion of the neuroprotective ectodomain sAPPαManuskript (preprint) (Annet vitenskapelig)
    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.

  • 266. Kratz, Felix
    et al.
    Senter, Peter
    Steinhagen, Henning
    Kurrikoff, Kaido
    Suhorutsenko, Julia
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-Penetrating Peptides in Cancer Targeting2012Inngår i: Drug Delivery in Oncology: From Basic Research to Cancer Therapy / [ed] Felix Kratz, Peter Senter, Henning Steinhagen, Wiley-VCH Verlagsgesellschaft, 2012, s. 1189-1217Kapittel i bok, del av antologi (Fagfellevurdert)
  • 267. Krolov, Katrin
    et al.
    Frolova, Jekaterina
    Tudoran, Oana
    Suhorutsenko, Julia
    Lehto, Taavi
    Sibul, Hiljar
    Mager, Imre
    laanpere, MaDe
    Tulp, Indrek
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples2014Inngår i: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 16, nr 1, s. 127-135Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds of women and therefore often goes undiagnosed. C. trachomatis infections are a major public health concern because of the potential severe long-term consequences, including an increased risk of ectopic pregnancy, chronic pelvic pain, and infertility. To date, several point-of-care tests have been developed for C. trachomatis diagnostics. Although many of them are fast and specific, they lack the required sensitivity for large-scale application. We describe a rapid and sensitive form of detection directly from urine samples. The assay uses recombinase polymerase amplification and has a minimum detection limit of 5 to 12 pathogens per test. Furthermore, it enables detection within 20 minutes directly from urine samples without DNA purification before the amplification reaction. Initial analysis of the assay from clinical patient samples had a specificity of 100% (95% CI, 92%-100%) and a sensitivity of 83% (95% CI, 51%-97%). The whole procedure is fairly simple and does not require specific machinery, making it potentially applicable in point-of-care settings.

  • 268. Krõlov, Katrin
    et al.
    Uusna, Julia
    Grellier, Tiia
    Andresen, Liis
    Jevtuševskaja, Jekaterina
    Tulp, Indrek
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings2017Inngår i: Expert Review of Molecular Diagnostics, ISSN 1473-7159, E-ISSN 1744-8352, Vol. 17, nr 12, s. 1117-1125Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: A variety of sample preparation techniques are used prior to nucleic acid amplification. However, their efficiency is not always sufficient and nucleic acid purification remains the preferred method for template preparation. Purification is difficult and costly to apply in point-of-care (POC) settings and there is a strong need for more robust, rapid, and efficient biological sample preparation techniques in molecular diagnostics. Methods: Here, the authors applied antimicrobial peptides (AMPs) for urine sample preparation prior to isothermal loop-mediated amplification (LAMP). AMPs bind to many microorganisms such as bacteria, fungi, protozoa and viruses causing disruption of their membrane integrity and facilitate nucleic acid release. Results: The authors show that incubation of E. coli with antimicrobial peptide cecropin P1 for 5 min had a significant effect on the availability of template DNA compared with untreated or even heat treated samples resulting in up to six times increase of the amplification efficiency. Conclusion: These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.

  • 269. Kurrikoff, Kaido
    et al.
    Gestin, Maxime
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Recent in vivo advances in cell-penetrating peptide-assisted drug delivery2016Inngår i: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 13, nr 3, s. 373-387Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Introduction: Delivery of macromolecular drugs is an important field in medical research. However, macromolecules are usually unable to cross the cell membrane without the assistance of a delivery system. Cell penetrating peptides (CPPs) are unique tools to gain access to the cell interior and deliver a bioactive cargo into the cytosol or nucleus. In addition to macromolecular delivery, CPPs have been used to deliver smaller bioactive molecules. Therefore CPPs have become an intensive field of research for medical treatment.

    Areas covered: In this review, we highlight studies that include CPP in vivo disease models. We review different strategies and approaches that have been used, with specific attention on recent publications. The approaches that have been used include CPP–cargo covalent conjugation strategies and nanoparticle strategies. Various additional strategies have been used to achieve disease targeting, including active targeting, passive targeting, and combined active/passive strategies. As a result, delivery of various types of molecule has been achieved, including small drug molecules, proteins and nucleic acid-based macromolecules (e.g. siRNA, antisense nucleotides and plasmid DNA).

    Expert Opinion: Despite recent advances in the field, confusions surrounding CPP internalization mechanisms and intracellular trafficking are hindering the development of new and more efficient vectors. Nevertheless, the recent increase in the number of publications containing in vivo CPP utilization looks promising that the number of clinical trials would also increase in the near future.

  • 270. Kurrikoff, Kaido
    et al.
    Veiman, Kadi-Liis
    Künnapuu, Kadri
    Peets, Elin Madli
    Lehto, Tõnis
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pärnaste, Ly
    Arukuusk, Piret
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Effective in vivo gene delivery with reduced toxicity, achieved by charge and fatty acid -modified cell penetrating peptide2017Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikkel-id 17056Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Non-viral gene delivery systems have gained considerable attention as a promising alternative to viral delivery to treat diseases associated with aberrant gene expression. However, regardless of extensive research, only a little is known about the parameters that underline in vivo use of the nanoparticle-based delivery vectors. The modest efficacy and low safety of non-viral delivery are the two central issues that need to be addressed. We have previously characterized an efficient cell penetrating peptide, PF14, for in vivo applications. In the current work, we first develop an optimized formulation of PF14/pDNA nanocomplexes, which allows removal of the side-effects without compromising the bioefficacy in vivo. Secondly, based on the physicochemical complex formation studies and biological efficacy assessments, we develop a series of PF14 modifications with altered charge and fatty acid content. We show that with an optimal combination of overall charge and hydrophobicity in the peptide backbone, in vivo gene delivery can be augmented. Further combined with the safe formulation, systemic gene delivery lacking any side effects can be achieved.

  • 271. Kurrikoff, Kaido
    et al.
    Veiman, Kadi-Liis
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Tartu University, Estonia.
    CPP-Based Delivery System for In Vivo Gene Delivery2015Inngår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, Vol. 1324, s. 339-347Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    The method presents most important steps in estimating a CPP-mediated reporter gene delivery in a mouse model. The method is applicable for administrating noncovalent CPP/pDNA complexes via i.v. injection and for analysis of luciferase levels in tissue homogenates. This method could be extended to analyze the delivery of different nucleic acid cargos with other types of delivery vectors. First, a simple method is presented for assessing the stability of complexes in blood after i.v. administration, based on quantitation of a fluorescently labeled nucleic acid. Secondly, a protocol is presented for assessing and analyzing luciferase levels in mouse organs.

  • 272. Kuteeva, Eugenia
    et al.
    Wardi, Tara
    Lundström, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sollenberg, Ulla
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hökfelt, Tomas
    Ögren, Sven Ove
    Differential Role of Galanin Receptors in the Regulation of Depression-Like Behavior and Monoamine/Stress-Related Genes at the Cell Body Level2008Inngår i: Neuropsychopharmacology, ISSN 0893-133X, E-ISSN 1740-634X, Vol. 33, nr 11, s. 2573-2585Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The present study on rat examined the role of galanin receptor subtypes in regulation of depression-like behavior as well as potential molecular mechanisms involved in the locus coeruleus (LC) and dorsal raphe (DR). The effect of intracerebroventricular (i.c.v.) infusion of galanin or galanin receptor GalR1- and GalR2-selective ligands was studied in the forced swim test, followed by quantitative in situ hybridization studies. Naive control, non-treated (swim control), saline-and fluoxetine-treated rats were used as controls in the behavioral and in situ hybridization studies. Subchronic treatment with fluoxetine reduced immobility and climbing time. Intracerebroventricular infusion of galanin, the GalR1 agonist M617 or the GalR2 antagonist M871 increased, while the GalR2(R3) agonist AR-M1896 decreased, immobility time compared to the aCSF-treated animals. Galanin also decreased the time of climbing. Galanin mRNA levels were upregulated by the combination of injection + swim stress in the saline-and the fluoxetine-treated groups in the LC, but not in the DR. Also tyrosine hydroxylase levels in the LC were increased following injection + swim stress in the saline-and fluoxetine-treated rats. Tryptophan hydroxylase 2 and serotonin transporter mRNAs were not significantly affected by any treatment. 5-HT(1A) mRNA levels were downregulated following i.c.v. galanin, M617 or AR-M1896 infusion. These results indicate a differential role of galanin receptor subtypes in depression-like behavior in rodents: GalR1 subtype may mediate 'prodepressive' and GalR2 'antidepressant' effects of galanin. Galanin has a role in behavioral adaptation to stressful events involving changes of molecules important for noradrenaline and/or serotonin transmission.

  • 273. Laettig-Tuennemann, Gisela
    et al.
    Prinz, Manuel
    Hoffmann, Daniel
    Behlke, Joachim
    Palm-Apergi, Caroline
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Morano, Ingo
    Herce, Henry D.
    Cardoso, M. Cristina
    Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides2011Inngår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 2, s. 453-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.

  • 274. Laffray, Sophie
    et al.
    Bouali-Benazzouz, Rabia
    Papon, Marie-Amelie
    Favereaux, Alexandre
    Jiang, Yang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holm, Tina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Spriet, Corentin
    Desbarats, Pascal
    Fossat, Pascal
    Le Feuvre, Yves
    Decossas, Marion
    Heliot, Laurent
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Nagy, Frederic
    Landry, Marc
    Impairment of GABAB receptor dimer by endogenous 14-3-3 zeta in chronic pain conditions2012Inngår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, nr 15, s. 3239-3251Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3 zeta, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3 zeta is overexpressed and weakens GABAB inhibition. Using anti-14-3-3 zeta siRNA or competing peptides disrupts 14-3-3 zeta/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization. The EMBO Journal (2012) 31, 3239-3251. doi:10.1038/emboj.2012.161; Published online 12 June 2012

  • 275.
    Langel, Kent
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Copolovici, Dana
    Arukuusk, Piret
    Sillard, Rannar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Novel fatty acid modifications of transportan 102010Inngår i: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 16, nr 4, s. 247-255Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are able toefficiently internalize into cells and can therefore be usedas vectors for non-viral cellular delivery of different cargoes.Previous studies have shown that hydrophobicmodifications of different CPPs can increase their transfectionefficiency dramatically. In this study we havemodified the cell penetrating-peptide transportan 10 (TP10)with a variety of hydrophobic molecules to determine therole of hydrophobicity in the uptake of these molecules.The results can be used to synthesize more efficientdelivery vectors. To evaluate how these constructs are ableto transport cargoes into cells we used 20-OMe splicecorrecting oligonucleotides. Non-covalent peptide-cargocomplexes were formed and their transfection efficiencywas measured using a luciferase readout system. Thehydrophobicity of the novel modifications was correlatedwith their biological efficacy. We determined the mostefficient range of hydrophobicity for TP10 analogs fordelivering oligonucleotides into cells. In order to assesshow the transfection efficacy of these particles is dependenton their size the hydrodynamic diameter of the formednanoparticles was measured using dynamic light scattering.These findings will be used to develop highly efficient nonviralgene therapy vectors

  • 276.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-Penetrating Peptides: Methods and Protocols2011Collection/Antologi (Fagfellevurdert)
  • 277.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-Penetrating Peptides: Preface.2015Inngår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, Vol. 1324, s. v-viiiKapittel i bok, del av antologi (Annet vitenskapelig)
  • 278.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Handbook of cell-penetrating peptides2007Collection/Antologi (Fagfellevurdert)
  • 279.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Preface2011Inngår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ûlo Langel, Humana Press, 2011Kapittel i bok, del av antologi (Annet vitenskapelig)
  • 280.
    Langel, Ülo
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cravatt, Benjamin F.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Land, Tiit
    Niessen, Sherry
    Zorko, Matjaz
    Introduction to peptides and proteins2010 (oppl. 1)Bok (Fagfellevurdert)
  • 281. Larsson, Emilia
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Raffalli-Mathieu, Françoise
    Quantitative and selective polymerase chain reaction analysis of highly similar human alpha-class glutathione transferases2011Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 412, nr 1, s. 96-101Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Alpha-class glutathione transferases (GSTs) found expressed in human tissues constitute a family of four homologous enzymes with contrasting enzyme activities. In particular, GST A3-3 has been shown to contribute to the biosynthesis of steroid hormones in human cells and is selectively expressed in steroidogenic tissues. The more ubiquitous GST A1-1, GST A2-2, and GST A4-4 appear to be primarily involved in detoxification processes and are expressed at higher levels than GST A3-3. We are interested in studying the cell and tissue expression of the GST A3-3 gene, yet the existence of highly expressed sequence-similar homologs and of several splice variants is a serious challenge for the specific detection of unique transcript species. We found that published polymerase chain reaction (PCR) primers for GST A3-3 lack the specificity required for reliable quantitative analysis. Therefore, we designed quantitative PCR (qPCR) primers with greatly increased discrimination power for the human GSTA3 full-length transcript. The improved primers allow accurate discrimination between GST A3-3 and the other alpha-class GSTs and so are of great value to studies of the expression of the GSTA3 gene. The novel primers were used to quantify GSTA3 transcripts in human embryonic liver and steroidogenic cell lines.

  • 282.
    Larsson, Veronica J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The roles of inner nuclear membrane proteins during interphase and mitosis2014Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The nuclear envelope (NE) consists of two concentric membranes, the outer nuclear membrane (ONM) and the inner nuclear membrane (INM). The LINC (linker of nucleoskeleton and cytoskeleton) complex spans both the ONM and the INM connecting the cytoskeleton to the nucleoskeleton and chromatin. Only a few of the known INM proteins have been functionally characterized and shown to have important roles in chromatin organisation. Defects in the genes coding for proteins in the INM and the nuclear lamina give rise to serious human diseases, called envelopathies.

    In 2009 (Buch et al. 2009) our group made two major discoveries. We showed for the first time, that an integral INM protein distributed along the microtubules of the mitotic spindle. This protein was therefore named Samp1, Spindle Associated Membrane Protein 1. The second discovery was that depletion of Samp1 caused detachment of the centrosome from the NE, suggesting that Samp1 is associated with the microtubule cytoskeleton both in interphase and mitosis.

    In this thesis we continued to investigate Samp1´s role during interphase. We also wanted to investigate the localisation of Samp1 in the mitotic spindle and possible function during mitosis. We show that the expression of Samp1 mutants and depletion of Samp1 affects the distribution and organisation of A-type lamins, the LINC complex protein Sun1 and the LINC complex associated protein emerin. Thus, in interphase Samp1 is functionally connected to the LINC complex and the A-type lamina network. The LINC complex can help explain how the centrosomes detach from the NE in Samp1 depleted cells. In mitotic cells, we found that depletion of Samp1 caused prolonged metaphase and aberrant mitotic phenotypes such as bi-nucleation, enlarged nuclei and micronuclei. We also showed that Samp1 interacts with RanGTPase and importin-β, which are key players in assembling the mitotic spindle. Samp1 also modulates the levels of importin-β and NuMA in the mitotic spindle, which could explain the mitotic phenotypes that we se in Samp1 depleted cells. Here we present evidence showing, for the first time, that an INM protein is present on kinetochore microtubules and have an essential role for correct chromosome segregation and spindle assembly.

  • 283.
    Larsson, Veronica J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jafferali, Mohammed Hakim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Markus, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The integral nuclear membrane protein Samp1 is essential for the mitotic process by modulating the levels of importin-β and NuMA in the mitotic spindleManuskript (preprint) (Annet vitenskapelig)
  • 284.
    Larsson, Veronica J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jafferali, Mohammed Hakim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Markus, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The integral nuclear membrane protein, Samp1 modulates importin-β and NuMA in the mitotic spindleManuskript (preprint) (Annet vitenskapelig)
  • 285.
    Larsson, Veronica J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jafferali, Mohammed Hakim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Vijayaraghavan, Balaje
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mitotic spindle assembly and correct chromosome segregation depend on the integral nuclear membrane protein, Samp1Manuskript (preprint) (Annet vitenskapelig)
  • 286.
    Larsson, Veronica J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jafferali, Mohammed Hakim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Vijayaraghavan, Balaje
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein, Samp12018Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 131, nr 8, artikkel-id jcs211664Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have investigated a possible role of the inner nuclear membrane protein, Samp1, in the mitotic machinery. Live cell imaging showed that Samp1aYFP distributed as filamentous structures in the mitotic spindle, partially co-localising with ß-tubulin. Samp1 depletion resulted in an increased frequency of cells with signs of chromosomal mis-segregation and prolonged metaphase, indicating problems with spindle assembly and/or chromosomal alignment. Consistently, mitotic spindles in Samp1 depleted cells contained significantly lower levels of ß-tubulin and γ-tubulin, phenotypes which were rescued by overexpression of Samp1aYFP. We found that Samp1 can bind directly to γ-tubulin and that Samp1 co-precipitated with γ-tubulin and HAUS6 of the Augmin complex in live cells. The levels of Haus6, in the mitotic spindle also decreased after Samp1 depletion. We show that Samp1 is involved in the recruitment of Haus6 and γ-tubulin to the mitotic spindle. Samp1 is the first inner nuclear membrane protein shown to have a function in mitotic spindle assembly.

  • 287. Le Rouzic, Erwann
    et al.
    Mousnier, Aurlie
    Rustum, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Stutz, Françoise
    Hallberg, Einar
    Dargemont, Catherine
    Benichou, Serge
    Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG12002Inngår i: J. Biol. Chem., ISSN 0021-9258, Vol. 277, nr 47, s. 45091-45098Artikkel i tidsskrift (Fagfellevurdert)
  • 288.
    Lehto, Taavi
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Abes, Rachida
    Oskolkov, Nikita
    Suhorutšenko, Julia
    Copolovici, Dana-Maria
    Mäger, Imre
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Viola, Joana R.
    Simonson, Oscar E.
    Ezzat, Kariem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eriste, Elo
    Smith, Edvard
    Lebleu, Bernard
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy2010Inngår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 141, nr 1, s. 42-51Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)4 peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)4 peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine™ 2000. We show that stearyl-(RxR)4 mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)4 or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine™ 2000, we show that stearyl-(RxR)4 is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)4 complexed with 2′-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)4 promotes dose-dependent splice correction in parity with (RxR)4-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)4 an intriguing vector for future in vivo experiments.

  • 289. Lehto, Taavi
    et al.
    Ezzat, Kariem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Peptide Nanoparticles for Oligonucleotide Delivery2011Inngår i: Progress in Molecular Biology and Translational Science: Nanoparticles in Translational Science and Medicine / [ed] Villaverde, A., Elsevier Academic Press , 2011, s. 397-426Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    In the past two decades, different methods have emerged for intervention with gene expression, which can be generally referred to as gene therapy. Oligonucleotides (ONs) and their analogs form the basis of the molecules that can be used to modulate gene expression. Unfortunately, due to their physicochemical properties, these molecules require assistance in their intracellular delivery. Cell-penetrating peptides (CPPs) are one class of nonviral delivery vectors that, because of their remarkable translocation properties, have been intensely utilized for the delivery, of ON-based molecules, both in vitro and in vivo. This chapter concentrates on the applications of CPPs that directly form nanopartides with different ONs and facilitate their intracellular delivery.

  • 290. Lehto, Taavi
    et al.
    Kurrikoff, Kaido
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Cell-penetrating peptides for the delivery of nucleic acids2012Inngår i: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 9, nr 7, s. 823-836Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Introduction: Different gene therapy approaches have gained extensive interest lately and, after many initial hurdles, several promising approaches have reached to the clinics. Successful implementation of gene therapy is heavily relying on finding efficient measures to deliver genetic material to cells. Recently, non-viral delivery of nucleic acids and their analogs has gained significant interest. Among non-viral vectors, cell-penetrating peptides (CPPs) have been extensively used for the delivery of nucleic acids both in vitro and in vivo. Areas covered: In this review we will discuss recent advances of CPP-mediated delivery of nucleic acid-based cargo, concentrating on the delivery of plasmid DNA, splice-correcting ONs, and small-interfering RNAs. Expert opinion: CPPs have proved their potential as carriers for nucleic acids. However, similarly to other non-viral vectors, CPPs require further development, as efficient systemic delivery is still seldom achieved. To achieve this, CPPs should be modified with entities that would allow better endosomal escape, targeting of specific tissues and cells, and shielding agents that increase the half-life of the vehicles. Finally, to understand the clinical potential of CPPs, they require more thorough investigations in clinically relevant disease models and in pre-clinical and clinical studies.

  • 291.
    Lehto, Taavi
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Simonson, Oscar E.
    Mäger, Imre
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ezzat, Kariem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sork, Helena
    Copolovici, Dana-Maria
    Viola, Joana R.
    Zaghloul, Eman M.
    Lundin, Per
    Moreno, Pedro M. D.
    Mäe, Maarja
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oskolkov, Nikita
    Suhorutšenko, Julia
    Smith, C. I. Edvard
    EL Andaloussi, Samir
    A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo2011Inngår i: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 19, nr 8, s. 1457-1467Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.

  • 292.
    Lehto, Tõnis
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Vasconcelos, Luis
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Margus, Helerin
    Figueroa, Ricardo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pooga, Margus
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides2017Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 28, nr 3, s. 782-792Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well studied CPP, PepFectl4, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.

  • 293.
    Lehto, Tönis
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Acylated cell-penetrating peptides for nucleic acid delivery2017Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In recent decades many new methods have been developed to cure or treat genetical disorders such as cancer, viral infections or inheritable diseases. The problem is that the nucleic acids and their synthetic analogs, oligonucleotides, are not able to cross the cell membrane due to their physicochemical properties like high negative charge and size. Therefore they need assistance to reach their intracellular target.

    Cell-penetrating peptides (CPPs) are a class of versatile delivery vectors that can be used to transport various types of bioactive molecules inside the cells, including proteins, small molecules and also nucleic acids like plasmid DNA (pDNA), splice-correcting oligonucleotides (SCO), small interfering RNA (siRNA) and messenger RNA (mRNA).

    A well-known method to improve CPPs in non-covalent delivery of nucleic acids is to modify them N-terminally with fatty acids such as stearic acid (C18:0). In this thesis we have studied the role of N-terminal acylation and the length of the carbon chain in the delivery of short SCO as well as larger plasmid DNA. In paper I we varied the N-terminal acyl chain length of a well-studied stearylated CPP, PepFect14, from 2-22 carbons. The delivery efficiency of SCO was dependent on the acyl chain length and it was found to be proportional to the increased association of peptide/oligonucleotide complexes to the cell membrane. In paper II the versatility of PepFect14 as a non-covalent nucleic acid delivery vector was validated using plasmid DNA. Compared to its non-stearylated counterpart, PepFect14 was able to condense pDNA into stable nanoparticles and mediate high gene expression both in regular adherent cell lines as well as difficult-to-transfect primary cells.

  • 294. Leist, Marcel
    et al.
    Ghallab, Ahmed
    Graepel, Rabea
    Marchan, Rosemarie
    Hassan, Reham
    Hougaard Bennekou, Susanne
    Limonciel, Alice
    Vinken, Mathieu
    Schildknecht, Stefan
    Waldmann, Tanja
    Danen, Erik
    van Ravenzwaay, Ben
    Kamp, Hennicke
    Gardner, Iain
    Godoy, Patricio
    Bois, Frederic Y.
    Braeuning, Albert
    Reif, Raymond
    Oesch, Franz
    Drasdo, Dirk
    Höhme, Stefan
    Schwarz, Michael
    Hartung, Thomas
    Braunbeck, Thomas
    Beltman, Joost
    Vrieling, Harry
    Sanz, Ferran
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swetox, Karolinska Institutet, Sweden.
    Gadaleta, Domenico
    Fisher, Ciaran
    Kelm, Jens
    Fluri, David
    Ecker, Gerhard
    Zdrazil, Barbara
    Terron, Andrea
    Jennings, Paul
    van der Burg, Bart
    Dooley, Steven
    Meijer, Annemarie H.
    Willighagen, Egon
    Martens, Marvin
    Evelo, Chris
    Mombelli, Enrico
    Taboureau, Olivier
    Mantovani, Alberto
    Hardy, Barry
    Koch, Bjorn
    Escher, Sylvia
    van Thriel, Christoph
    Cadenas, Cristina
    Kroese, D.
    van de Water, Bob
    Hengstler, Jan G.
    Adverse outcome pathways: opportunities, limitations and open questions2017Inngår i: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 91, nr 11, s. 3477-3505Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event erelationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

  • 295.
    Lilja, Johanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Development of a sensory neuronal cell model for the estimation of mild eye irritation.2004Inngår i: Altern Lab Anim, ISSN 0261-1929, Vol. 32, nr 4, s. 339-43Artikkel i tidsskrift (Annet vitenskapelig)
  • 296.
    Lilja, Johanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Laulund, Frida
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin and Insulin-Like Growth FactorType-I Up-Regulate the Vanilloid Receptor-1(TRPV1) in Stably TRPV1-ExpressingSH-SY5Y Neuroblastoma Cells2007Inngår i: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 85, nr 7, s. 1413-1419Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The capsaicin receptor, transient receptor potential, vanilloid type 1 (TRPV1), is a Ca2+ permeable ion channel activated by noxious stimuli eliciting pain. Several reports have shown modulation of TRPV1 activity and expression by neuronal growth factors. Here, we study the long-term effects on TRPV1 expression mediated by insulin like growth factor type-I (IGF-I) and insulin in a stably TRPV1-expressing SH-SY5Y neuroblastoma cell line. We show that after 72 h of 10 nM IGF-I or insulin exposure, the TRPV1 protein level was up-regulated 2.5 and 2-fold, respectively. By blocking phosphatidylinositol-3-kinase (PI(3)K) or mitogen-activated protein kinase (MAPK) signaling we concluded that the increase in total TRPV1 protein content induced by IGF-I was controlled by PI(3)K signaling whereas insulin seemed to regulate TRPV1 protein expression via both PI(3)K and MAPK pathways. Inhibiting protein kinase C (PKC) blocked the effects of both IGF-I and insulin. Furthermore, the concentrations causing 50 % Ca2+ increase (EC50) after insulin and IGF-I-treatments were significantly lowered compared to untreated cells. We conclude that IGF-I and insulin enhance TRPV1 protein expression and activity, and impaired pain sensation might result from distorted TRPV1 regulation in the peripheral nervous system.

  • 297.
    Lilja, Johanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindegren, Helene
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Surfactant-Induced TRPV1 Activity—A Novel Mechanismfor Eye Irritation?2007Inngår i: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 99, nr 1, s. 174-180Artikkel i tidsskrift (Fagfellevurdert)
    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. 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+ influxes 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 eye irritating products.

  • 298.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides for oligonucleotide delivery: Design and uptake mechanisms2014Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The use of oligonucleotides for gene therapy has the potential to efficiently treat a plethora of diseases with minimal side effects. However, the use of oligonucleotides is hampered by the properties of these molecules, which make it essentially impossible for them to permeate the cellular membrane. Therefore, a great deal of research has been focused on developing delivery vectors, which can efficiently and safely deliver oligonucleotides into cells. Cell-penetrating peptides (CPPs) constitute a class of delivery vectors that have received much attention since they were discovered over 20 years ago. CPPs can deliver a wide variety of cargos into cells, such as small molecules, proteins, oligonucleotides and particles, in an efficacious and non-toxic manner.

    In this thesis two new CPPs for oligonucleotide delivery were designed. The purpose of the design was to create CPPs, which form stable complexes with oligonucleotides and have endosomolytic properties. The new peptides showed superior potency in intracellular oligonucleotide delivery compared to previously reported CPPs. These results demonstrate that it is possible to drastically improve the efficiency of existing CPPs with relatively simple modifications.

    It is well known that CPPs use endocytosis to gain entry into cells, however, why cells endocytose CPPs has never been clearly established. In this thesis we determine that several CPP:oligonucleotide complexes interact with scavenger receptors, and that this interaction leads to endocytosis. The results presented in this thesis provides a deeper understanding of how CPPs function and thereby insights how to improve CPP design.

  • 299.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Rational design of novel cell-penetrating peptides2012Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Although cell-penetrating peptides (CPPs) have been an active field of research for over 20 years there are still properties such as efficiency and stability that must be improved for CPPs to reach their full potential as delivery vectors.

    In this thesis two peptides were designed for non-covalent oligonucleotide delivery, in an attempt to overcome two major issues hampering wider use of CPPs, namely low stability and endosomal entrapment.

    In Paper I we designed a new peptide named PepFect14, which has unnatural amino-acid ornithine as the source of positive charge, to address the stability issue. PepFect14 was shown to deliver SCOs in a highly efficient manner into different cell-lines. Furthermore, formulation of non-covalent PepFect14:SCO complexes into solid form, suitable for storage and transportation, was developed. The formulated complexes were stable for weeks and retained high activity.

    In Paper II we modified PepFect14 with endosomolytic groups to facilitate endosomal escape, the resulting peptide was named PepFect15. Delivery of SCO and miRNA was efficiently mediated by PepFect15 into HeLa cells and endosomolytic property was validated. Additionally, uptake of Pepfect15:SCO complexes was shown to be mediated by scavenger receptor class A.

  • 300.
    Lindberg, Staffan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Muñoz-Alarcón, Andrés
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Helmfors, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mosqueira, Diogo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gyllborg, Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tudoran, Oana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors2012Inngår i: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 441, nr 1-2, s. 242-247Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Gene-regulatory biomolecules such as splice-correcting oligonucleotides and anti-microRNA oligonucleotides are important tools in the struggle to understand and treat genetic disorders caused by defective gene expression or aberrant splicing. However, oligonucleotides generally suffer from low bioavailability, hence requiring efficient and non-toxic delivery vectors to reach their targets. Cell-penetrating peptides constitute a promising category of carrier molecules for intracellular delivery of bioactive cargo. In this study we present a novel cell-penetrating peptide, PepFect15, comprising the previously reported PepFect14 peptide modified with endosomolytic trifluoromethylquinoline moieties to facilitate endosomal escape. Pepfect15 efficiently delivers both splice-correcting oligonucleotides and anti-microRNA oligonucleotides into cells through a non-covalent complexation strategy. To our knowledge this is the first work that describes peptide-mediated anti-microRNA delivery. The peptide and its cargo form stable, negatively charged nanoparticles that are taken up by cells largely through scavenger receptor type A mediated endocytosis.

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