Einfluss der endo-lysosomalen Qb-SNAREs Vti1a und Vti1b auf die subzelluläre Sortierung sowie auf signalgebende Prozesse im zentralen Nervensystem der Maus
Creators: name: Bollmann, Christian
Publisher: name: Universität Bielefeld
Year: 2024
Subjects: subject: 572

description: The last step in intracellular transport of eukaryotic cells is the fusion of two opposing membranes which is mediated by SNARE proteins forming a four-helix bundle to provide the driving force necessary for fusion. The Qb-SNAREs Vti1a and Vti1b are known to be implicated in several endo-lysosomal fusion processes. In mice, the single knockout of either Vti1a or Vti1b is associated with only minor phenotypic alterations, whereas the double knockout of both SNAREs results in perinatal lethality. Vti1a/Vti1b-double deficient embryos show several phenotypic abnormalities, including severe developmental defects of the central nervous system (CNS). In contrast Vti1a/Vti1b-double heterozygous mice do not show an obvious phenotype. <br><br>

In the present study, a subcellular analysis of the organelle structure revealed strong Golgi abnormalities in Vti1a-/- Vti1b-/- neurons. In vitro, the Golgi in hippocampal Vti1a-/- Vti1b-/- neurons showed a compact morphology and was mostly concentrated in the perinuclear region of the cell, whereas the Golgi of respective control cells was widely distributed throughout the soma and typically extended into the first segment of one or two dendrites. Furthermore, discrete Golgi structures (Golgi outposts) were absent in the dendritic processes of Vti1a-/- Vti1b-/- neurons. An altered Golgi morphology was also evident in pyramidal cells of the hippocampal CA1 subfield in situ, as demonstrated by immunohistological examination of Vti1a/Vti1b-double deficient embryonic brain tissue sections. However, an analysis of the ER showed no striking alterations in Vti1a-/- Vti1b-/-neurons.
Since earlier investigations have already demonstrated an impaired TGF-β-/BMP-dependent signaling in Vti1a-/- Vti1b-/- chondrocytes, the functionality of the associated signaling pathways in Vti1a-/- Vti1b-/- neurons were analyzed. However, no defects were detected here. Following BMP-2 stimulation, cultured Vti1a/Vti1b-double deficient neurons exhibited a similar increase in the level of Smad phosphorylation as respective controls. Likewise, the nuclear translocation of Smad1 induced by BMP-2 was not provably influenced in Vti1a-/- Vti1b-/- neurons. Furthermore, the activity of the signaling pathways in the central nervous system of Vti1a-/- Vti1b-/- embryos was examined. No remarkable impairments were found here as well. Thus, in cortical, hippocampal and cerebellar tissues the pathway activity of both Smad1/5/9- and Smad2-dependent signaling was not altered due to the loss of Vti1a and Vti1b. 
At the postsynaptic side of excitatory synapses, neurotransmitter receptors are attached to the cytoskeleton via a dense protein network known as postsynaptic density (PSD), which clusters the receptors within the postsynaptic membrane and couples their activation to several intracellular signaling events. Using protein biochemical analysis, an altered composition of the PSD was detected in Vti1a-/- Vti1b-/- embryos. The amount of the ionotropic AMPA-receptor was increased in the PSD of Vti1a-/- Vti1b-/- embryos compared to controls, whereas the postsynaptic level of PSD-95, an important anchor protein of the PSD, was slightly reduced. A subsequent analysis of central signaling pathways revealed a markedly reduced activity of the MAPK signaling pathway in neuronal tissue of the CNS of Vti1a-/- Vti1b-/- embryos. In contrast, the activity of the Akt signaling pathway as well as the signaling via CaMKII was not altered in case of Vti1a/Vti1b-double deficiency. These initial results suggest a possible link between an altered composition of the PSD and an influenced signaling in the central nervous system of Vti1a-/- Vti1b-/- embryos. <br>
Finally, a neuronal Vti1a-knockout cell line was successfully generated using the CRISPR/Cas9n-technique. The knockout of Vti1a was validated by different methods on protein, transcript and genomic level. A subsequent knockout of Vti1b will lead to a Vti1a/Vti1b-double deficient cell line, which will be used as a complementary model system to elucidate the functional role of Vti1a and Vti1b with respect to the neuronal background.