Analysis of mice deficient in late endosomal SNARE proteins VAMP8/endobrevin and Vti1b

Abstract

Membrane fusion requires a synchronized interplay between several proteins that form the membrane fusion machinery. SNAREs are the central component of this fusion machinery. Cognate SNARE proteins localized to transport vesicles and their target membranes interact with each other via coiled-coil interaction to form core complexes. Vesicle associated membrane protein-8 (VAMP8) or endobrevin is a mammalian R-SNARE that participates in the formation of two complexes. First, VAMP8 in a complex with syntaxin8, syntaxin7 and Vti1b, mediates the homotypic late endosomal fusion. Second, VAMP8 forms a complex with SNAP23 and syntaxin 4 and mediates exocytosis of zymogen granules in the pancreatic acinar cells. VAMP8 is also required for exocytosis of granules from mast cells and platelets. This study was aimed at understanding the physiological role of VAMP8 using VAMP8 knock out mice. VAMP8-/- mice were normal at birth, however, by 10-12 days of age nearly one thirds of the mice were dead. These one third mice started to loose weight at postnatal day 8-9 (small not sick stage) and after 2-3 consecutive days of weight loss (small and sick stage), the mice died. The survivors became healthy adults but were lighter than littermates. The small and sick VAMP8-/- mice were half the weight of the littermates and had an extremely small thymus. The thymic medulla was reduced to mere remnants and there was no clear cortico-medullary boundary. The developing thymocytes including the CD4-CD8- DN1-4 subsets and CD4/CD8 populations showed a major maturational disturbance. The thymus of small and sick VAMP8-/- mice showed high number of dead cells and the thymocytes from small not sick VAMP8-/- mice were highly sensitive to apoptotic stimuli in vitro. Bone marrow derived hemopoietic progenitors from small and sick VAMP8-/- mice could develop into functional T and B lymphocytes in the RAG2 -/- γc-/- mice, indicating that the thymic stromal cells could be defective in the VAMP8-/- mice but the thymocyte precursors are normal. Hence, VAMP8 is important in proper development and function of thymus in mice and the loss of VAMP8 has severe implications on the thymocytes and the thymic stroma. However processes such as endocytosis, endosomal traffic, phagocytosis and lysosomal degradation are not affected in the cells derived from VAMP8-/- mice, indicating that VAMP8 is probably not essential for the late endosomal fusion events.