Role of SNARE proteins and endosomal transport in survival and differentiation of neurons
Final Report Abstract
In eukaryotic cells, membrane-enclosed organelles communicate by vesicular traffic. Membrane trafficking pathways are classified into a biosynthetic/exocytotic limb and an endocytotic/degradative limb that originates at the plasma membrane and leads via endosomes to lysosomes. Evolutionarily conserved trafficking steps are adapted to generate specialized membrane compartments in neurons. Each step involves formation of transport vesicles by budding, transport of vesicles to their destination, attachment and subsequent fusion with the target membrane. Transport is carried out by members of protein families, which are conserved between unicellular eukaryotes and man. SNAREs catalyze membrane fusion. In this project we studied mice deficient for the SNAREs vti1a (localized to the trans Golgi network and early endosomes) and vti1b (localized to early endodosomes, late endosomes and lysosomes). As Vti1a-/- Vti1b-/- (DKO) fibroblasts survive with intact organelle morphology and minor trafficking defects we tried to identify compensatory SNAREs. However, we were not able to find a clear answer. Hippocampal neurons from DKO embryos had shorter neurites in culture. On one hand, DKO neurons were less responsive to various neurotrophic factors than control neurons. On the other hand, the enlargesome pathway, which is one of several pathways contributing membranes during neurite outgrowth, was affected in DKO neurons. The neuroendocrine chromaffin cells contain chromaffin granules, which are large dense core vesicles undergoing regulated secretion. Vti1a-/- chromaffin cells had less and smaller chromaffin granules indicating that biogenesis of chromaffin granules is influenced by vti1a. Vti1b-/- chromaffin cells were not affected. The defect was not exacerbated in Vti1a-/- Vti1b-/- chromaffin cells. These data indicate that vti1b cannot substitute for vti1a in chromaffin granule biogenesis. Vti1a had also a specific role in recycling of synaptic vesicle proteins after exocytosis. Finally, DKO embryos displayed abnormalities in their skeletal system, which are reminiscent of phenotypes of Tgf-β2-/- and Bmp-7-/- mice. Ligands of the TGF-ß and BMP-family are important regulators of bone formation. We found that TGF-ß1 and BMP-2 signaling is impaired in DKO chondrocytes suggesting that mislocalization of their receptors could contribute to the skeletal phenotypes. Cell migration is also important in bone formation and reduced in DKO fibroblasts. This project benefitted tremendously from the purchase of a confocal microscope in May 2013, which was financed as a large instrument by DFG and the state Nordrhein-Westfalen.
Publications
- (2014) The SNARE protein vti1a functions in dense‐core vesicle biogenesis. EMBO J. 33, 1681-1697
Walter, AM., Kurps, J., de Wit, H., Schöning, S., Toft‐Bertelsen, TL., Lauks, J., Ziomkiewicz, I., Weiss, AN., Schulz, A., Fischer von Mollard, G., Verhage, M., Sørensen, JB.
(See online at https://doi.org/10.15252/embj.201387549)
