Doctor of Philosophy

dissertationLipid membranes organize eukaryotic cells into functional compartments called organelles. Material is delivered to and from organelles in a regulated fashion. Vesicles bud from a source compartment, move across the cell and fuse with a target membrane. SNARE proteins, with Sec1/Munc18 (SM) proteins, drive the fusion of vesicles with their target by bridging the apposing membranes and forcing them together. The SNARE/SM fusion complex is essential for all vesicle fusion. Each trafficking pathway utilizes a different set of SNARE/SM family members. In the nervous system the secretory pathway is responsible for the release of neurotransmitters, which pass signals between neurons. The neuronal SNAREs include synaptobrevin, syntaxin, and SNAP-25. However, it is not clear that these are the only SNAREs responsible for neurotransmitter release. In fact countless studies have reported residual neurotransmission in the absence of each of these proteins, raising the question what is the mechanism responsible for residual fusion in neuronal SNARE knockouts? In Chapter 2, I explore this question by focusing on the neuronal SNARE SNAP-25. We characterize the snap-25 genetic locus in C. elegans and examine the physiology of neurons lacking the SNAP-25 protein. We find that SNAP-25 plays an important role in docking and fusing synaptic vesicles but is not strictly essential for either one. We reveal that the conserved SNARE protein, SNAP-29 is capable of substituting for SNAP-25 in synaptic vesicle fusion. We demonstrate that the SNAP-29 protein is natively expressed in neurons and localized at synapses. Our observations suggest that the canonical neuronal SNAREs may not act alone in releasing neurotransmitters. Finally, I explore the mechanism by which the neuronal SM protein (Unc18) facilitates fusion. Unc18 binds SNAREs in three configurations. A binary complex with syntaxin is important for trafficking. At nerve terminals, UNC-18 interacts with an N-terminal peptide on syntaxin and with the SNARE four-helix bundle. Our experiments demonstrate that the N-peptide of syntaxin is a passive tether facilitating Unc18's transition from the binary syntaxin interaction to a direct interaction with the ternary SNARE complex. Future work is required to elucidate the fusogenic properties of Unc18's interaction with the ternary complex

MN1 C-terminal truncation syndrome is a novel neurodevelopmental and craniofacial disorder with partial rhombencephalosynapsis

MN1 encodes a transcriptional co-regulator without homology to other proteins, previously implicated in acute myeloid leukaemia and development of the palate. Large deletions encompassing MN1 have been reported in individuals with variable neurodevelopmental anomalies and non-specific facial features. We identified a cluster of de novo truncating mutations in MN1 in a cohort of 23 individuals with strikingly similar dysmorphic facial features, especially midface hypoplasia, and intellectual disability with severe expressive language delay. Imaging revealed an atypical form of rhombencephalosynapsis, a distinctive brain malformation characterized by partial or complete loss of the cerebellar vermis with fusion of the cerebellar hemispheres, in 8/10 individuals. Rhombencephalosynapsis has no previously known definitive genetic or environmental causes. Other frequent features included perisylvian polymicrogyria, abnormal posterior clinoid processes and persistent trigeminal artery. MN1 is encoded by only two exons. All mutations, including the recurrent variant p.Arg1295* observed in 8/21 probands, fall in the terminal exon or the extreme 3' region of exon 1, and are therefore predicted to result in escape from nonsense-mediated mRNA decay. This was confirmed in fibroblasts from three individuals. We propose that the condition described here, MN1 C-terminal truncation (MCTT) syndrome, is not due to MN1 haploinsufficiency but rather is the result of dominantly acting C-terminally truncated MN1 protein. Our data show that MN1 plays a critical role in human craniofacial and brain development, and opens the door to understanding the biological mechanisms underlying rhombencephalosynapsis.status: publishe