![]() ![]() We first performed two-color coexpression experiments and examined the ability of the Munc18-1 C180Y mutant to incorporate Munc18-1 WT proteins into large aggregates by two-color single-molecule detection ( Martin et al., 2014 Fig. As widespread aggregation was observed in Munc18-1 C180Y-expressing cells, we questioned whether these could contribute to a dominant pathological function by incorporating the WT Munc18-1 protein. These experiments revealed the existence of large aggregates of Munc18-1 C180Y, as indicated by the detection of bright particles, detected as large fluctuations in fluorescence intensity that were never observed for Munc18-1 WT molecules ( Martin et al., 2014). We also examined the process of Munc18-1 C180Y aggregation using single-molecule fluorescence spectroscopy to directly assess oligomerization of fluorescently tagged Munc18-1 WT and Munc18-1 C180Y expressed in a eukaryotic-cell-free system ( Martin et al., 2014). The mutation promotes misfolding and aggregation, potentiating the protein’s ubiquitination and degradation by the proteasome, thereby inhibiting its availability for membrane fusion ( Saitsu et al., 2008 Martin et al., 2014). 1 A) leads to its cellular aggregation, with catastrophic effects on neuroexocytosis ( Martin et al., 2014). In a recent study, we demonstrated that expression of Munc18-1 carrying the missense EIEE4-causing mutation (C180Y Fig. In this study, we reveal a critical new role for Munc18-1 in chaperoning α-synuclein (α-Syn), thereby controlling its aggregative propensity and ability to form toxic α-Syn oligomers. In addition, dysregulation of Munc18-1 expression has been associated with other neurological disorders, including Alzheimer’s disease ( Jacobs et al., 2006 Donovan et al., 2012) and Rasmussen encephalitis ( Alvarez-Barón et al., 2008). The mechanism by which these mutations lead to the pathological epileptic phenotype is poorly understood and may involve either haploinsufficiency of the wild-type (WT) protein or a gain of pathological function of the mutant allele. Munc18-1 mutations linked to the development of EIEE4 include intragenic and whole-gene deletions, as well as 10 different missense mutations ( Saitsu et al., 2008, 2010 Otsuka et al., 2010). EIEEs comprise a group of rare but severe developmental disorders, with a poor initial prognosis (50% of patients die within the first year Tavyev Asher and Scaglia, 2012 Barcia et al., 2014). Recently, however, Munc18-1 heterozygous mutations have been associated with several developmental diseases, including nonsyndromic intellectual disability, epilepsy ( Hamdan et al., 2009, 2011 Deprez et al., 2010 Otsuka et al., 2010 Mignot et al., 2011), and early infantile epileptic encephalopathy (EIEE Saitsu et al., 2008, 2010). A milestone study demonstrated that Munc18-1 knockout leads to perinatal paralysis-induced lethality but, importantly, does not affect brain development ( Verhage et al., 2000). It acts by transporting a SNARE protein called syntaxin1A to the plasma membrane ( Han et al., 2009 Malintan et al., 2009 Martin et al., 2013 Papadopulos et al., 2013) and by regulating the formation of the SNARE complex during synaptic vesicle priming, a process that drives the fusion of synaptic vesicles, thereby mediating neurotransmitter release at the synapse. Munc18-1 is an essential component of the molecular machinery that controls SNARE-mediated membrane fusion in neurons and neuroendocrine cells. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration. Coexpression of the α-Syn A30P mutant with Munc18-1 reduced the number of α-Syn A30P aggregates. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-Syn WT and that of the Parkinson’s disease–causing α-Syn A30P mutant, an effect rescued by Munc18-1 WT expression, indicative of chaperone activity. We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. ![]()
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