Reduction in Synaptic Vesicle Protein Abundance but Increased Amounts of Nsg2 and Lpcat1 in Cerebral Cortices Without the Endosomal SNARE Proteins Vti1a and Vti1b

Absence of the endosomal SNAREs vti1a and vti1b results in perinatal death and severe neuronal phenotypes in mice, while lack of one of these proteins results in minor phenotypes. Proteomic differences were investigated to obtain a deeper insight into processes in which vti1a and vti1b are involved. Here we applied a bottom-up shotgun proteomic approach to investigate the differences in wild-type, double heterozygous (DHET), vti1a^-/- vti1b^-/- double knockout (DKO), vti1a^-/- knockout and vti1b^-/- knockout cerebral cortices. Single deletions did not affect protein levels significantly. A total of 1725 proteins were detected of which 69 were less abundant and 191 proteins were more abundant in DKO cortices. Many less abundant proteins belonged to cellular components and reactome pathways synapse, synaptic vesicle cycle, vesicle mediated transport, L1CAM interaction, and cholesterol biosynthesis in pathway enrichment analysis. More abundant proteins were enriched in cellular components and Kyoto Encyclopaedia of Genes and Genomes (KEGG)-pathways such as spliceosome, ribosome, carbon metabolism, and ribonucleoprotein complex. Immunoblotting validated reduced expression levels of the tested synaptic vesicle proteins as well as increased amounts of lysophosphatidylcholine acyltransferase 1 (Lpcat1) and neuron-specific gene 2 (Nsg2), which is involved in postsynaptic AMPA-receptor recycling. These data indicate that the synapse and cell adhesion were strongly affected in DKO brains. STATEMENT OF SIGNIFICANCE OF THE STUDY: Distinct populations of neurons and glia cells are generated and organize into layers during brain development. Neurons develop an elaborate morphology to transmit information via axons and synapses to dendrites in receiving neurons. These neurites form via several specialized pathways of vesicle secretion and endocytosis. Fusion between these membranes requires members of the SNARE protein family. Double knockouts of the endosomal SNAREs vti1a and vti1b (DKO) result in perinatal lethality in mice with massive defects in the brain. In this study we compared the proteome of DKO brain cortices with double heterozygous controls to obtain insights into the molecular alterations and affected pathways. DKO brains contained lower amounts of synaptic proteins and proteins involved in cell adhesion, membrane trafficking and cholesterol biosynthesis. Several proteins of spliceosomes, ribosomes and carbon metabolism were more abundant in DKO brains, which may be a consequence of the reduced amounts of synaptic proteins or a shift in cell populations. Lysophosphatidylcholine acyltransferase 1 (Lpcat1) and neuron-specific gene 2 (Nsg2), which is involved in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling, were confirmed to be more abundant by Western blotting. These data point to defects in trafficking especially in the synapse and in cell adhesion, which is required for neurite outgrowth.