Primary neurons lacking the SNAREs vti1a and vti1b show altered neuronal development.

BACKGROUND

Neurons are highly specialized cells with a complex morphology generated by various membrane trafficking steps. They contain Golgi outposts in dendrites, which are formed from somatic Golgi tubules. In trafficking membrane fusion is mediated by a specific combination of SNARE proteins. A functional SNARE complex contains four different helices, one from each SNARE subfamily (R-, Qa, Qb and Qc). Loss of the two Qb SNAREs vti1a and vti1b from the Golgi apparatus and endosomes leads to death at birth in mice with massive neurodegeneration in peripheral ganglia and defective axon tracts.

METHODS

Hippocampal and cortical neurons were isolated from Vti1a Vti1b double deficient, Vti1a Vti1b, Vti1a Vti1b and Vti1a Vti1b double heterozygous embryos. Neurite outgrowth was determined in cortical neurons and after stimulation with several neurotrophic factors or the Rho-associated protein kinase ROCK inhibitor Y27632, which induces exocytosis of enlargeosomes, in hippocampal neurons. Moreover, postsynaptic densities were isolated from embryonic Vti1a Vti1b and Vti1a Vti1b control forebrains and analyzed by western blotting.

RESULTS

Golgi outposts were present in Vti1a Vti1b and Vti1a Vti1b dendrites of hippocampal neurons but not detected in the absence of vti1a and vti1b. The length of neurites was significantly shorter in double deficient cortical neurons. These defects were not observed in Vti1a Vti1b and Vti1a Vti1b neurons. NGF, BDNF, NT-3, GDNF or Y27632 as stimulator of enlargeosome secretion did not increase the neurite length in double deficient hippocampal neurons. Vti1a Vti1b postsynaptic densities contained similar amounts of scaffold proteins, AMPA receptors and NMDA receptors compared to Vti1a Vti1b, but much more TrkB, which is the receptor for BDNF.

CONCLUSION

The absence of Golgi outposts did not affect the amount of AMPA and NMDA receptors in postsynaptic densities. Even though TrkB was enriched, BDNF was not able to stimulate neurite elongation in Vti1a Vti1b neurons. Vti1a or vti1b function as the missing Qb-SNARE together with VAMP-4 (R-SNARE), syntaxin 16 (Qa-SNARE) and syntaxin 6 (Qc-SNARE) in induced neurite outgrowth. Our data show the importance of vti1a or vti1b for two pathways of neurite elongation.