The double deficiency of the SNARE proteins vti1a and vti1b affects neurite outgrowth and signaling in N1E-115 neuroblastoma cells.

Abstract

During intracellular trafficking N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins catalyze the membrane fusion by assembling into a four-helix complex. In the mouse model, loss of the endosomal SNAREs vti1a and vti1b results in a perinatal lethal phenotype and neuronal defects including decreased neurite outgrowth in cultured primary neurons.

We used a CRISPR/Cas9 system to generate a Vti1a Vti1b double knockout (DKO) in the neuroblastoma cell line N1E-115. Three different DKO cell lines were obtained and examined at genome and protein level. The double deficiency impaired proper differentiation based on lower levels of synaptic proteins as well as reduced neurite formation and elongation compared to wild type cells in differentiation medium. Neurite elongation can be induced by a variety of extracellular signals via different signaling cascades. Treatment with the Rho kinase inhibitor Y27632, which stimulates enlargeosome exocytosis, or with neurotrophic factors (BDNF, NGF and NT3) resulted in reduced stimulation of all DKO clones and in significantly shorter neurites compared to wild type cells. The loss of vti1a and vti1b disrupted Akt signaling during enlargeosome-mediated and Erk signaling during BDNF-induced neurite outgrowth.