Specification and induction of ontogenetic/programmed cell death through extrinsic signals
Final Report Abstract
Programmed cell death (PCD) is essential for the development of the nervous system and for its maintenance. While a significant amount of information is available on the intrinsic pathway(s) of apoptosis ranging from regulators and effectors to executioners, little is known on the induction and specification of neuron death during nervous system development. The goal of the project was to develop a better understanding of the extrinsic and intrinsic signals that specify and mediate ontogenetic cell death. The study was performed utilizing the best established model systems for PCD in the developing chick, ciliary ganglionic and lumbar motorneurons (MN) as the in vivo models. With the advent of the knowledge of the chick genome, the chick is now also suited to elucidate molecular determinants and design appropriate gain- and loss-of-function experiments. First, we performed a genome-wide screen for both neuron populations covering the execution time of cell death from E9 to E14. With the help of appropriate bioinformatics and statistics we generated a list of nineteen candidate genes for the PCD period for CG neurons, based on their differential expression and statistical power. For the analyses of their role within programmed cell death in vivo a shRNA-mediated knockdown approach was established. For the genes BID, ACVR2B and RARB for example the expression is upregulated prior to the execution of PCD. The present study has shown that after the shRNA-mediated knockdown of BID, ACVR2B and RARB-expression, the number of CG neurons dying during the time of PCD is reduced, while for the RNAi constructs targeting ACVR2B and RARB, even the differentiation of CG neurons is delayed. Nevertheless, while the knockdown of BID and ACVR2B permanently prevented PCD to take place, RARB knockdown exhibited a shift in the execution time of cell death from E9 to E14. The results of the RNAi experiments further corroborate the biological significance of this gene expression pattern. After the knockdown of ACVR2B expression, cell death was inhibited and neuronal differentiation was impaired. While the neuro differentiation defect might partly be explained by a lack of BMP signaling, this is the first study showing a role of ActRIIB-mediated signaling in neuronal cell death. Second, for the analyses of cell death of lumbar MNs, human small heat shock proteins (HSPBs) were selected as a candidate family of proteins. Mutations in HSPBs can lead to human disease and particularly point mutations in HSPB1 and HSPB8 are known to lead to peripheral neuropathies. Recently, a missense mutation (R7S) in HSPB3 was found to cause an axonal motor neuropathy (distal hereditary motor neuropathy type 2C). We studied the endogenous HSPB3 protein distribution in the spinal cords of chicken and mouse embryos and in the postnatal nervous system (central and peripheral) of chicken, mouse and human. We further investigated the impact of wild-type and mutated HSPB3 on MN cell death via overexpressing these genes in ovo in an avian model of MN degeneration, the limbbud removal. The quantification of the surviving MNs three days after the induction of degeneration showed that both HSPB3-WT and R7S were able to rescue MNs from cell death, albeit the latter less markedly than the former, and that they induced a similar cell loss when overexpressed in healthy MNs (without LBR). We therefore conclude that HSPB3 plays a significant role in spinal MNs survival.
Publications
- (2015) RARβ regulates neuronal cell death and differentiation in the avian ciliary ganglion. Dev Neurobiol. 75(11):1204-18
Koszinowski S, Boerries M, Busch H, Krieglstein K
(See online at https://doi.org/10.1002/dneu.22278) - (2015) Signaling via the transcriptionally regulated activin receptor 2B is a novel mediator of neuronal cell death during chicken ciliary ganglion development. Int J Dev Neurosci. 41:98-104
Koszinowski S, Buss K, Kaehlcke K, Krieglstein K
(See online at https://doi.org/10.1016/j.ijdevneu.2015.01.006) - (2015) The combination of limb-bud removal and in ovo electroporation techniques: a new powerful method to study gene function in motoneurons undergoing lesion-induced cell death. J Neurosci Methods. 239:206-13
La Padula V, Koszinowski S, Krieglstein K
(See online at https://doi.org/10.1016/j.jneumeth.2014.10.022) - (2016) HSPB3 protein is expressed in motoneurons and induces their survival after lesion-induced degeneration. Experimental Neurology
Volume 286, December 2016, Pages 40-49
La Padula V, Staszewski O, Nestel S; Busch H, Boerries M, Roussa E, Prinz M, Krieglstein K
(See online at https://doi.org/10.1016/j.expneurol.2016.08.014)
