Loss-of-function mutations in RAB18 cause Warburg micro syndrome.

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.

Structural and functional deficits in a neuronal calcium sensor-1 mutant identified in a case of autistic spectrum disorder.

Neuronal calcium sensor-1 (NCS-1) is a Ca(2+) sensor protein that has been implicated in the regulation of various aspects of neuronal development and neurotransmission. It exerts its effects through interactions with a range of target proteins one of which is interleukin receptor accessory protein like-1 (IL1RAPL1) protein. Mutations in IL1RAPL1 have recently been associated with autism spectrum disorders and a missense mutation (R102Q) on NCS-1 has been found in one individual with autism. We have examined the effect of this mutation on the structure and function of NCS-1. From use of NMR spectroscopy, it appeared that the R102Q affected the structure of the protein particularly with an increase in the extent of conformational exchange in the C-terminus of the protein. Despite this change NCS-1(R102Q) did not show changes in its affinity for Ca(2+) or binding to IL1RAPL1 and its intracellular localisation was unaffected. Assessment of NCS-1 dynamics indicated that it could rapidly cycle between cytosolic and membrane pools and that the cycling onto the plasma membrane was specifically changed in NCS-1(R102Q) with the loss of a Ca(2+) -dependent component. From these data we speculate that impairment of the normal cycling of NCS-1 by the R102Q mutation could have subtle effects on neuronal signalling and physiology in the developing and adult brain.

The functions of Munc18-1 in regulated exocytosis.

The activation of regulated exocytosis occurs by a rise in cytosolic Ca(2+) concentration. Synaptotagmins act as the Ca(2+) sensors, whereas the machinery that allows fusion of secretory vesicles with the plasma membrane consists of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including syntaxin 1, SNAP-25, and VAMP. Within the pathway leading to exocytosis, there is an essential requirement for a member of the conserved Sec1/Munc18 (SM) protein family, which in neurotransmitter and neurohormone release in mammalian cells is Munc18-1. The exact role of Munc18-1 and the steps within exocytosis in which it acts have been intensively investigated. Current evidence suggests that Munc18-1 acts via distinct modes of interactions with syntaxin 1 and the other SNARE proteins and influences all of the steps leading to exocytosis, including vesicle recruitment, tethering, docking, priming, and membrane fusion.

The Rab27 effector Rabphilin, unlike Granuphilin and Noc2, rapidly exchanges between secretory granules and cytosol in PC12 cells.

Rab proteins are GTPases that transit between GTP- and GDP-bound states. In the GTP-bound form they can recruit specific effector to membrane domains. It is possible that the exchange of Rab effectors between membranes and cytosol would be determined by the exchange of the particular Rab partner. We have compared the cycling of three Rab3/27 effectors, Granuphilin, Noc2, and Rabphilin, in PC12 cells using fluorescence recovery after photobleaching of EGFP-tagged proteins. All three effectors become localised to secretory granules. Granuphilin and Noc2 showed little or no exchange between secretory granules and cytosol whereas Rabphilin showed rapid and complete exchange. Both Noc2 and Rabphilin were found to be recruited to granules by Rab27 but the data suggest that Rabphilin did not form stable complexes with Rab27 on secretory granules and so Rab effector cycling between membranes and cytosol can be independent of that of the Rab protein.

A gain-of-function mutant of Munc18-1 stimulates secretory granule recruitment and exocytosis and reveals a direct interaction of Munc18-1 with Rab3.

Munc18-1 plays a crucial role in regulated exocytosis in neurons and neuroendocrine cells through modulation of vesicle docking and membrane fusion. The molecular basis for Munc18 function is still unclear, as are the links with Rabs and SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) receptor] proteins that are also required. Munc18-1 can bind to SNAREs through at least three modes of interaction, including binding to the closed conformation of syntaxin 1. Using a gain-of-function mutant of Munc18-1 (E466K), which is based on a mutation in the related yeast protein Sly1p, we have identified a direct interaction of Munc18-1 with Rab3A, which is increased by the mutation. Expression of Munc18-1 with the E466K mutation increased exocytosis in adrenal chromaffin cells and PC12 cells (pheochromocytoma cells) and was found to increase the density of secretory granules at the periphery of PC12 cells, suggesting a stimulatory effect on granule recruitment through docking or tethering. Both the increase in exocytosis and changes in granule distribution appear to require Munc18-1 E466K binding to the closed form of syntaxin 1, suggesting a role for this interaction in bridging Rab- and SNARE-mediated events in exocytosis.

Differential dynamics of Rab3A and Rab27A on secretory granules.

We have assessed the dynamics of the association of Rab3A and Rab27A with secretory granules at various stages of their life in PC12 cells. Endogenous Rab3A colocalised with the secretory granule marker secretogranin II (SGII) and expressed EGFP-Rab3A and ECFP-Rab27A colocalised with one another. The extent of colocalisation between EGFP-Rab3A or EGFP-Rab27 and SGII increased after longer times post transfection suggesting that these Rab proteins are preferentially recruited to newly synthesised granules. Following the release of immature secretory granules from the trans-Golgi network, Rab3A and Rab27A became associated with the immature granules after a lag period of around 20 minutes. Rab dynamics on granules were analysed in fluorescence recovery after photobleaching (FRAP) experiments. The recovery profile of EGFP-Rab27A was comparable to that of ppANF-EGFP, whereas the recovery profile of EGFP-Rab3A was significantly faster, indicating that Rab3A but not Rab27A might be rapidly exchanged between granules and cytosol. Inhibition of heat-shock protein 90 with 10 muM geldanamycin did not affect the exchange process or regulated exocytosis. Rab dynamics during stimulation with 300 muM ATP were analysed in live cells. Loss of granular ppANF-EGFP fluorescence was seen at the cell periphery after stimulation but only limited changes in EGFP-Rab3A and EGFP-Rab27A fluorescence was observed, indicating that the Rab proteins do not immediately dissociate or disperse on stimulation. The data suggest potentially distinct roles for Rab3A and Rab27A and we suggest that the finding that young secretory granules have a higher capacity for binding Rab3A and Rab27A is functionally important for preferential exocytosis from these granules.