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Alterations in receptor expression and function in scrapie-infected neuronal cell lines
Stockholm University, Faculty of Science, Department of Neurochemistry.
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: urn:nbn:se:su:diva-163ISBN: 91-7265-890-8 (print)OAI: oai:DiVA.org:su-163DiVA: diva2:190347
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
List of papers
1. Loss of lipopolysaccharide-induced nitric oxide production and inducible nitric oxide synthase expression in scrapie-infected N2a cells
Open this publication in new window or tab >>Loss of lipopolysaccharide-induced nitric oxide production and inducible nitric oxide synthase expression in scrapie-infected N2a cells
2003 (English)In: Journal of neuroscience research, ISSN 0360-4012, Vol. 71, no 2, 291-299 p.Article in journal (Refereed) Published
Abstract [en]

In scrapie-infected cells, the conversion of the cellular prion protein to the pathogenic prion has been shown to occur in lipid rafts, which are suggested to function as signal transduction platforms. Neuronal cells may respond to bacterial lipopolysaccharide (LPS) treatment with a sustained and elevated nitric oxide (NO) release. Because prions and the major LPS receptor CD14 are colocalized in lipid rafts, the LPS-induced NO production in scrapie-infected neuroblastoma cells was studied. This study shows that LPS induces a dose- and time-dependent increase in NO release in the murine neuroblastoma cell line N2a, with a 50-fold increase in NO production at 1 g/ml LPS after 96 hr, as measured by nitrite in the medium. This massive NO release was not caused by activation of the neuronal NO synthase (nNOS), but by increased expression of the inducible NOS (iNOS) mRNA and protein. 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 due not to abolished enzymatic activity of iNOS but to a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and RT-PCR. These results indicate that scrapie infection inhibits the LPS-mediated signal transduction upstream of the transcriptional step in the signaling cascade and may reflect the important molecular and cellular changes induced by scrapie infection

Place, publisher, year, edition, pages
Wiley Interscience, 2003
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-23136 (URN)10.1002/jnr.10473 (DOI)
Note
Part of urn:nbn:se:su:diva-163Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2009-12-21Bibliographically approved
2. Altered protein tyrosine phosphorylation in scrapie-infected neuronal cell lines
Open this publication in new window or tab >>Altered protein tyrosine phosphorylation in scrapie-infected neuronal cell lines
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23137 (URN)
Note
Part of urn:nbn:se:su:diva-163Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2010-01-13Bibliographically approved
3. 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: 2017-12-13Bibliographically approved
4. 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: 2017-12-13Bibliographically approved

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