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Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
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

Place, publisher, year, edition, pages
2001. Vol. 276, no 39, 36110-36115 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-23138OAI: oai:DiVA.org:su-23138DiVA: diva2:190345
Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2011-05-05Bibliographically approved
In thesis
1. Alterations in receptor expression and function in scrapie-infected neuronal cell lines
Open this publication in new window or tab >>Alterations in receptor expression and function in scrapie-infected neuronal cell lines
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This study shows that lipopolysaccharide (LPS) induces a robust, concentration-and time-dependent increase in nitric oxide (NO) production in the murine neuroblastoma cell line N2a by increased expression of iNOS. However, in scrapie-infected N2a cells (ScN2a), the LPS-induced NO production was completely abolished. The absence of LPS-induced NO production in ScN2a was not due to inhibited enzymatic activity of iNOS but a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and reverse transcription-polymerase chain reaction (RT-PCR).

Furthermore, major changes in the protein tyrosine phosphorylation profile of scrapie-infected hypothalamic neurons (ScGT1-1) and ScN2a, compared to their uninfected counterparts were shown, by Western blot of whole cell extracts and of anti-phosphotyrosine immunoprecipitates, with major bands corresponding to120, 150 and 180 kD. Anti-phosphotyrosine blots of lectin-purified glycoproteins, indicated that some, but not all, of the proteins in the 120 kD band are glycosylated. Two phosphoproteins of ≈ 120 kDa were identified, the receptor tyrosine kinase, fibroblast growth factor 2 (FGFR2) and the cytoplasmic non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (PYK2).

In addition, scrapie-infection induces important alterations in the expression, binding and signalling of two neurotrophic receptors, the insulin-like growth factor-1 receptor (IGF-1R) and the insulin receptor (IR) in ScN2a, as compared to uninfected N2a cells.

In ScN2a, the IGF-1R and IR protein levels were four- and two-fold increased, respectively, with an unexpected decrease in specific binding sites, as revealed by equilibrium binding studies. In the case of the IGF-1R, the apparent drop in binding sites was due to a seven-fold drop in IGF-1 affinity. Moreover, the IGF-1 stimulated IGF-1R tyrosine phosphorylation was increased in ScN2a when compared to the reduced affinity, possibly due to altered tyrosine kinase signalling in ScN2a. In the case of the IR, the binding affinity was unchanged, although insulin-stimulated IR tyrosine phosphorylation was increased.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2004. 116 p.
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-163 (URN)91-7265-890-8 (ISBN)
Public defence
2004-06-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Opponent
Supervisors
Available from: 2004-05-13 Created: 2004-05-13Bibliographically approved
2. Altered cell signaling linked to neurodegeneration: Studies on scrapie-infected neuroblastoma cells and activated microglia 
Open this publication in new window or tab >>Altered cell signaling linked to neurodegeneration: Studies on scrapie-infected neuroblastoma cells and activated microglia 
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
Prion disease, Inflammation, MAPK, Microglia activation, Neuroblastoma
National Category
Neurosciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-57222 (URN)978-91-7447-285-1 (ISBN)
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

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