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

• Published in: European journal of cell biology 2024-12, Vol.103 (4), p.151461, Article 151461
• Main Authors: Kotschnew, Katharina, Winkler, Denise, Reckmann, Jonas, Mann, Charlotte, Schweigert, Alina, Tellkamp, Greta, Müller, Kristian M., Fischer von Mollard, Gabriele
• Format: Article
• Language: English
• Subjects: Animals; Cell Line, Tumor; Enlargeosome; Mice; N1E-115; Neurite outgrowth; Neurites - metabolism; Neuroblastoma - genetics; Neuroblastoma - metabolism; Neuroblastoma - pathology; Neuronal Outgrowth; Neurotrophin; Qb-SNARE Proteins - genetics; Qb-SNARE Proteins - metabolism; Signal Transduction; SNARE; Vti1
• ISSN: 0171-9335; 1618-1298; 1618-1298
• DOI: 10.1016/j.ejcb.2024.151461

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. •N1E-115 cells are a useful model system for analyzing neuronal differentiation•Vti1a and Vti1b double knockout is generated in a single step using CRISPR/Cas9•Lack of the endosomal SNAREs vti1a and vti1b impair neuronal differentiation•Enlargeosome mediated neurite outgrowth and Akt signalling is decreased in double KO•Neurotrophin-induced neurite outgrowth and Erk signalling is reduced in double KO