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Experimental Studies of BMP Signalling in Neuronal Cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated.

This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2003. , p. 75
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1259
Keywords [en]
Neurosciences, Bone Morphogenetic Protein (BMP), Growth Differentiation Factor 10 (GDF10), Aktivne-receptor Like Kinase 2 (ALK2), Tyrosine Hydroxylase (TH), Neurotrophic Factor, Neurite Outgrowth, Hippocampus, Catecholamine, PC12, Sympathetic Ganglia, Substantia Nigra (SN), Gene Targeting
Keywords [sv]
Neurovetenskap
National Category
Neurology
Research subject
Developmental Neurosciences
Identifiers
URN: urn:nbn:se:uu:diva-3398ISBN: 91-554-5624-3 (print)OAI: oai:DiVA.org:uu-3398DiVA, id: diva2:162656
Public defence
2003-05-16, B21, BMC, Uppsala, 09:15
Opponent
Supervisors
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2013-03-22Bibliographically approved
List of papers
1. Targeted Deletion of GDF10 has no Effect on Long Term Potentiation, Contextual Learning Ability or Gene Transcription in the Hippocampus
Open this publication in new window or tab >>Targeted Deletion of GDF10 has no Effect on Long Term Potentiation, Contextual Learning Ability or Gene Transcription in the Hippocampus
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90335 (URN)
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2010-01-13Bibliographically approved
2. Normal Nigrostriatal Innervation but Dopamine Dysfunction in Mice Carrying Hypomorphic Tyrosine Hydroxylase Alleles
Open this publication in new window or tab >>Normal Nigrostriatal Innervation but Dopamine Dysfunction in Mice Carrying Hypomorphic Tyrosine Hydroxylase Alleles
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2003 (English)In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 72, no 4, p. 444-453Article in journal (Refereed) Published
Abstract [en]

We investigated the use of the mouse tyrosine hydroxylase (TH) gene to drive knock-in constructs in catecholaminergic neurons. Two targeting constructs representing truncated forms of either of the BMP receptors ALK-2 or BMPR-II preceded by an internal ribosome entry site (IRES) were introduced into the 3' untranslated region of TH. An frt-flanked neomycin-resistance (neo(r)) cassette was placed in the 3' end of the targeting constructs. Mice homozygous for the knock-in alleles showed various degrees of hypokinetic behavior, depending mainly on whether the neo(r) cassette was removed. In situ hybridization and immunohistochemistry showed that TH mRNA and protein were variously down-regulated in these mouse strains. Reduced levels of dopamine and noradrenalin were found in several brain areas. However, number and morphology of neurons in substantia nigra and their projections to striatum appeared normal in the neo(r)-positive TH hypomorphic mice as examined by markers for L-aromatic amino acid decarboxylase and the dopamine transporter. Elimination of the neo(r) cassette from the knock-in alleles partially restored TH and dopamine levels. The present neo(r)-positive TH hypomorphic mice show that nigrostriatal innervation develops independently of TH and should find use as a model for conditions of reduced catecholamine synthesis, as seen in, for example, L-dihydroxyphenylalanine-responsive dystonia/infantile parkinsonism.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-91750 (URN)10.1002/jnr.10606 (DOI)12704806 (PubMedID)
Note

De två första författarna delar första författarskapet.

Available from: 2004-04-21 Created: 2004-04-21 Last updated: 2017-12-14Bibliographically approved
3. Blocked MAP kinase activity selectively enhances neurotrophic growth responses
Open this publication in new window or tab >>Blocked MAP kinase activity selectively enhances neurotrophic growth responses
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2004 (English)In: Molecular and Cellular Neuroscience, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 25, no 2, p. 345-354Article in journal (Refereed) Published
Abstract [en]

Bone morphogenetic proteins (BMPs) 4 and 6 as well as MEK inhibitors PD98059 and U0126 potentiate neurotrophin 3 (NT3)- and neurturin (NTN)-induced neurite outgrowth and survival of peripheral neurons from the E9 chicken embryo. Preexposure to BMP4 or PD98059 was sufficient to prime the potentiation of subsequently added NT3. Phosphorylation of Erk2, induced by NT3, was reduced by MEK inhibition but unaffected by BMP signaling. Real-time PCR showed that neither BMP stimulation nor MEK inhibition increased Trk receptor expression and that the BMP-induced genes Smad6 and Id1 were not upregulated by PD98059. In contrast, both MEK inhibition and BMP signaling suppressed transcription of the serum-response element (SRE)-driven Egr1 gene. A reporter assay using NGF-stimulated PC12 cells demonstrated that MEK/Erk/Elk-driven transcriptional activity was inhibited by Smad1/5 and by PD98059. Thus, suppression of SRE-controlled transcription represents a likely convergence point for pathways regulating neurotrophic responses.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-90337 (URN)10.1016/j.mcn.2003.10.015 (DOI)15019950 (PubMedID)
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2017-12-14Bibliographically approved
4. Bone morphogenetic protein signalling in NGF-stimulated PC12 cells
Open this publication in new window or tab >>Bone morphogenetic protein signalling in NGF-stimulated PC12 cells
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2003 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 307, no 3, p. 632-639Article in journal (Refereed) Published
Abstract [en]

Bone morphogenetic proteins (BMPs) are shown to potentiate NGF-induced neuronal differentiation in PC12 phaeochromocytoma cells grown on collagen under low-serum conditions. Whereas, cell bodies remained rounded in control medium or with only BMPs present, addition of BMP4 or BMP6 robustly increased the neuritogenic effect of NGF within 2 days. NGF-increased phosphorylation of p44(Erk1) and p42(Erk2) between 2 and 24h was unaffected by addition of BMP6. PC12 cells transfected with the SBE(4x)-luc reporter showed that BMP4 significantly increased receptor-activated Smad activity. Expression of constitutively active BMP receptor ALK2 activating Smad1 and Smad5 resulted in a strong increase in the SBE(4x)-luc reporter response. Adding the inhibitory Smad7 drastically reduced this signal. In contrast to wild-type (wt) Smad5, a Smad5 variant lacking five Erk phosphorylation sites in the linker region (designated Smad5/5SA) showed a strong background transcriptional activity. A fusion construct (Gal4-Smad5/5SA) was also highly transcriptionally active. Addition of the MEK inhibitor U0126 to PC12 cells expressing Gal4-Smad5/wt did not increase background transcriptional activity. However, upon activation by constitutively active ALK2 both Gal4-Smad5/wt and Gal4-Smad5/5SA strongly stimulated transcription. The data show that serine residues of the linker region of Smad5 reduce spontaneous transcriptional activity and that NGF-activated Erk does not antagonise BMP signalling at this site. Hence, NGF and BMP signals are likely to interact further downstream at the transcriptional level in neuronal differentiation of the PC12 cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-90338 (URN)10.1016/S0006-291X(03)01236-1 (DOI)12893270 (PubMedID)
Available from: 2003-04-24 Created: 2003-04-24 Last updated: 2017-12-14Bibliographically approved

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