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Altered cell signaling linked to neurodegeneration: Studies on scrapie-infected neuroblastoma cells and activated microglia 
Stockholm University, Faculty of Science, Department of Neurochemistry.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Prion diseases are neurodegenerative disorders that can affect humans and animals. The underlying event is a conformational change of the normal cellular prion protein (PrPC) into an aberrant isoform termed PrP-scrapie (PrPSc). PrPSc is thought to lead to neurodegeneration and activation of glial cells.

Scrapie infection of neuroblastoma cells was shown to increase the expression of insulin receptor (IR). Additionally, a marked reduction of 125I-insulin binding sites was observed. Insulin stimulation showed alteration in both IR β-subunit tyrosine phosphorylation and extracellular signal regulated kinase-2 (ERK2) activity.  Furthermore, scrapie infection was shown to increase insulin-like growth factor-1(IGF-1) receptor (IGF-1R) expression, although the number of 125I-IGF-1-binding sites was reduced. Also binding affinity of 125I-IGF-1 to its receptor was reduced, and tyrosine phosphorylation of IGF-1R-β-subunit in response to IGF-1 was altered. The increased levels of neurotrophic receptors might represent a neuroprotective response to prion infection. However, scrapie infection instead leads to decreased function, decreased levels of functional receptors, or both, which could promote neurodegeneration in prion diseases, through attenuated neurotrophic support.

In BV-2 microglial cells, LPS-induced iNOS (inducible nitric oxide synthase) expression and subsequent NO production were mainly mediated through c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway. Antioxidant treatment indicates that oxidative suppressing mechanism(s) acts on JNK pathway possibly as a regulatory mechanism controlling the NO levels. The JNK pathway was also shown to play an important role in the survival of BV-2 cells. We show that BV-2 cells are protected from ongoing apoptosis by pro-survival activity mediated both by the JNK and p38 MAPK pathway during LPS-induced inflammation. This is very interesting findings since it is important for microglia to respond properly to a pathogen, without themselves being affected and undergo apoptosis.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2011. , 58 p.
Keyword [en]
Prion disease, Inflammation, MAPK, Microglia activation, Neuroblastoma
National Category
Neurosciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
URN: urn:nbn:se:su:diva-57222ISBN: 978-91-7447-285-1OAI: oai:DiVA.org:su-57222DiVA: diva2:414683
Public defence
2011-06-09, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2011-05-12 Created: 2011-05-04 Last updated: 2011-05-26Bibliographically approved
List of papers
1. Altered insulin receptor processing and function in scrapie-infected neuroblastoma cell lines
Open this publication in new window or tab >>Altered insulin receptor processing and function in scrapie-infected neuroblastoma cell lines
2001 (English)In: Brain Research. Molecular Brain Research, ISSN 0169-328X, E-ISSN 1872-6941, Vol. 97, no 2, 161-170 p.Article in journal (Refereed) Published
Abstract [en]

The underlying neurochemical changes contributing to prion-induced neurodegeneration remain largely unknown. This study showsthat scrapie infection induced a 2-fold increase of insulin receptor (IR) protein and aberrantly processed IR b-chain in scrapie-infectedN2a neuroblastoma cells (ScN2a) as measured byWestern blot of immunoprecipitated IR, in the absence of increased IR mRNA. ElevatedIR protein level was further confirmed in an independently scrapie-infected neuroblastoma cell line N1E-115 (ScN1E-115). Proliferationstudies showed that the increased IR level in ScN2a did not result in an increased insulin-mediated cell growth compared to normal N2a125 cells. Binding studies indicated that this apparent paradox was due to a 65% decrease in specific [ I]insulin binding sites in ScN2a whencompared to the amount of immunoreactive IR, although the IR binding affinity was unchanged. Analysis of insulin stimulated IR tyrosinephosphorylation showed a slight but not significant reduction in ScN2a, when related to the increased level of immunoreactive IR.However, comparing the IR tyrosine phosphorylation to the loss of binding sites in ScN2a, we demonstrated an increased IR tyrosinephosphorylation of the remaining functional IR. In addition to these differences in IR properties, the basal extracellular signal regulatedkinase-2 (ERK2) phosphorylation detected by Western blot, was significantly elevated and the insulin stimulated ERK2 phosphorylationwas subsequently decreased in ScN2a. Together, these data show that scrapie infection affects the level and processing of the IR andsignal transduction mediated by the IR in neuroblastoma cells, as well as induces an elevated basal ERK2 phosphorylation. Aberrantregulation of neuroprotective receptors may contribute to neurodegeneration in prion diseases.

Place, publisher, year, edition, pages
Elsevier, 2001
Keyword
Scrapie; Prion; Insulin receptor; Neuroblastoma and proliferation
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-23139 (URN)10.1016/S0169-328X(01)00316-3 (DOI)
Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2011-05-05Bibliographically approved
2. Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells
Open this publication in new window or tab >>Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells
2001 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 39, 36110-36115 p.Article in journal (Refereed) Published
Abstract [en]

A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie-infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in the level of IGF-1R in two independently scrapie-infected neuroblastomas, ScN2a and ScN1E-115 cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In contrast to the elevated IGF-IR expression in ScN2a, receptor binding studies revealed an 80% decrease in specific I-125-IGF-1-binding sites compared with N2a cells. This decrease in IGF-1R-binding sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1 stimulated IGF-1R beta tyrosine phosphorylation showed no major change in the dose-response between the cell types, possibly due to altered tyrosine kinase signaling in scrapie-infected neuroblastoma cells. Altogether these data indicate that scrapie infection affects the expression, binding affinity, and signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and function may weaken the trophic support in scrapie-infected neurons and thereby contribute to neurodegeneration in prion diseases

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-23138 (URN)
Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2011-05-05Bibliographically approved
3. LPS-induced iNOS expression in Bv-2 cells is suppressed by an oxidative mechanism acting on the JNK pathway-A potential role for neuroprotection
Open this publication in new window or tab >>LPS-induced iNOS expression in Bv-2 cells is suppressed by an oxidative mechanism acting on the JNK pathway-A potential role for neuroprotection
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2010 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 1322, no March 2010, 1-7 p.Article in journal (Refereed) Published
Abstract [en]

Activated microglia cells, observed during chronic inflammation, produce and secrete compounds that at high concentrations or during sustained production might cause neuronal cell death. Inducible nitric oxide synthase (iNOS) is expressed in response to various immunological stimuli and catalyses the formation of the free radical nitric oxide (NO), that at low and regulated levels participate in cell signaling and cytoprotective events, whereas its higher and unregulated production can promote neurotoxicity in cells or in tissues. Regulation of NO production is therefore central for maintaining NO-levels within a safe window. We have analyzed iNOS protein expression and NO production, in murine microglial Bv-2 cells after 16 h treatment with the bacterial endotoxin lipopolysaccharide (LPS). We have further analyzed three MAPK pathways, by co-treating the cells with LPS and the inhibitors of ERK1/2, p38 or JNK MAPK activities. To investigate participation of an oxidative regulatory mechanism, cells were also treated with the antioxidant N-acetyl-L-cysteine (NAC). Our results show that LPS-induced iNOS expression in Bv-2 cells is mainly mediated through JNK MAPK. In addition, co-treatment of the Bv-2 cells with LPS and NAC surprisingly further increased the iNOS expression, an effect also found to be mediated through the JNK MAPK pathway. The level of phosphorylated JNK MAPK (p46) was strongly increased by LPS alone and was further increased when combined with NAC. Our data indicate that iNOS and NO production are suppressed by an oxidative mechanism acting on the JNK MAPK pathway and we speculate that it might constitute a potential regulatory mechanism controlling the NO level.

Keyword
Microglial activation, Inflammation, Signal transduction pathways, NAC, ROS (Reactive oxygen species)
National Category
Neurosciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-50134 (URN)10.1016/j.brainres.2010.01.082 (DOI)000276621800001 ()
Note
authorCount :5Available from: 2010-12-21 Created: 2010-12-21 Last updated: 2011-05-05Bibliographically approved
4. Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells
Open this publication in new window or tab >>Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells
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2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 406, no 3, 488-92 p.Article in journal (Refereed) Published
Abstract [en]

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.

National Category
Natural Sciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-56125 (URN)10.1016/j.bbrc.2011.02.083 (DOI)000288973300033 ()21338578 (PubMedID)
Available from: 2011-04-08 Created: 2011-04-08 Last updated: 2012-01-19Bibliographically approved

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