Experimental Studies of BMP Signalling in Neuronal Cells

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 transforming growth factor beta superfamily
Smad as an intracellular signalling mediator
BMP target genes
Biological actions of BMPs
Neurotrophic factors
The family of neurotrophins
Neurotrophin induced intracellular signalling pathways
The GDNF family
Nerve cells examined
Catecholaminergic neurons
Present investigation
Paper I
Paper II
Paper III
Paper IV
Knockout without phenotypical alterations
Dopamine dysfunction in TH-hypomorphic mice
Concluding remarks
Paper I
Paper II
Paper III
Paper IV
Materials and methods
Targeted gene disruption/alteration (Paper I and II)
Generation of truncated ALK2 and BMPRII by PCR
Screening for genomic DNA clones and targeting vector construction
Culturing of embryonic stem (ES) cells
DNA-preparation and screening of ES-cell clones
Blastocyst injection and germ line transmission
Housing and Breeding
Tail biopsies and DNA-preparation
Behavioural testing (Paper I and II)
A protocol for primary phenotypic screening
Spontaneous movement (Paper I and II)
Spatial learning ability (Paper I)
Electrophysiological recordings (Paper I)
LTP in the CA1-region of the hippocampus and the medial lateral perforant paths of the dentate gyrus
Biological assays (Paper III)
Neurite outgrowth assays
Survival assay
Pharmacological inhibitors of MAPK pathways (Paper III)
Biochemical and molecular studies
Determination of catecholamine and 5HT levels (Paper II)
Immunofluorescence (Paper II)
In situ hybridisation (paper I and II)
RNA preparation and microarray analysis (Paper I)
Western blot (Papers III and IV)
Cell culture
PC12 cells
Luciferase assay (Papers II and IV)
Crosslinking (Paper II, supplementary data

Author: Althini, Susanna

Source: Uppsala University Library

Download URL 2: Visit Now

Leave a Comment