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.

Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi.

The SNAREs Vti1a/1b are implicated in regulated secretion, but their role relative to canonical exocytic SNAREs remains elusive. Here, we show that synaptic vesicle and dense-core vesicle (DCV) secretion is indeed severely impaired in Vti1a/b-deficient neurons. The synaptic levels of proteins that mediate secretion were reduced, down to 50% for the exocytic SNARE SNAP25. The delivery of SNAP25 and DCV-cargo into axons was decreased and these molecules accumulated in the Golgi. These defects were rescued by either Vti1a or Vti1b expression. Distended Golgi cisternae and clear vacuoles were observed in Vti1a/b-deficient neurons. The normal non-homogeneous distribution of DCV-cargo inside the Golgi was lost. Cargo trafficking out of, but not into the Golgi, was impaired. Finally, retrograde Cholera Toxin trafficking, but not Sortilin/Sorcs1 distribution, was compromised. We conclude that Vti1a/b support regulated secretion by sorting secretory cargo and synaptic secretion machinery components at the Golgi.