Trophic support delays but does not prevent cell-intrinsic degeneration of neurons deficient for munc18-1

The stability of neuronal networks is thought to depend on synaptic transmission which provides activity-dependent maintenance signals for both synapses and neurons. Here, we tested the relationship between presynaptic secretion and neuronal maintenance using munc18-1-null mutant mice as a model. These mutants have a specific defect in secretion from synaptic and large dense-cored vesicles [Verhage et al. (2000), Science, 287, 864-869; Voets et al. (2001), Neuron, 31, 581-591]. Neuronal networks in these mutants develop normally up to synapse formation but eventually degenerate. The proposed relationship between secretion and neuronal maintenance was tested in low-density and organotypic cultures and, in vivo, by conditional cell-specific inactivation of the munc18-1 gene. Dissociated munc18-1-deficient neurons died within 4 days in vitro (DIV). Application of trophic factors, insulin or BDNF delayed degeneration up to 7 DIV. In organotypic cultures, munc18-1-deficient neurons survived until 9 DIV. On glial feeders, these neurons survived up to 10 DIV and 14 DIV when insulin was applied. Co-culturing dissociated mutant neurons with wild-type neurons did not prolong survival beyond 4 DIV, but coculturing mutant slices with wild-type slices prolonged survival up to 19 DIV. Cell-specific deletion of munc18-1 expression in cerebellar Purkinje cells in vivo resulted in the specific loss of these neurons without affecting connected or surrounding neurons. Together, these data allow three conclusions. First, the lack of synaptic activity cannot explain the degeneration in munc18-1-null mutants. Second, trophic support delays but cannot prevent degeneration. Third, a cell-intrinsic yet unknown function of munc18-1 is essential for prolonged survival

Analysis of Sec22p in Endoplasmic Reticulum/Golgi Transport Reveals Cellular Redundancy in SNARE Protein Function

Membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form heteromeric complexes that are required for intracellular membrane fusion and are proposed to encode compartmental specificity. In yeast, the R-SNARE protein Sec22p acts in transport between the endoplasmic reticulum (ER) and Golgi compartments but is not essential for cell growth. Other SNARE proteins that function in association with Sec22p (i.e., Sed5p, Bos1p, and Bet1p) are essential, leading us to question how transport through the early secretory pathway is sustained in the absence of Sec22p. In wild-type strains, we show that Sec22p is directly required for fusion of ER-derived vesicles with Golgi acceptor membranes. In sec22Δ strains, Ykt6p, a related R-SNARE protein that operates in later stages of the secretory pathway, is up-regulated and functionally substitutes for Sec22p. In vivo combination of the sec22Δ mutation with a conditional ykt6-1 allele results in lethality, consistent with a redundant mechanism. Our data indicate that the requirements for specific SNARE proteins in intracellular membrane fusion are less stringent than appreciated and suggest that combinatorial mechanisms using both upstream-targeting elements and SNARE proteins are required to maintain an essential level of compartmental organization