SummaryMutations in the Golgi SNARE (SNAP [soluble NSF attachment protein] receptor) protein Membrin (encoded by the GOSR2 gene) cause progressive myoclonus epilepsy (PME). Membrin is a ubiquitous and essential protein mediating ER-to-Golgi membrane fusion. Thus, it is unclear how mutations in Membrin result in a disorder restricted to the nervous system. Here, we use a multi-layered strategy to elucidate the consequences of Membrin mutations from protein to neuron. We show that the pathogenic mutations cause partial reductions in SNARE-mediated membrane fusion. Importantly, these alterations were sufficient to profoundly impair dendritic growth in Drosophila models of GOSR2-PME. Furthermore, we show that Membrin mutations cause fragmentation of the presynaptic cytoskeleton coupled with transsynaptic instability and hyperactive neurotransmission. Our study highlights how dendritic growth is vulnerable even to subtle secretory pathway deficits, uncovers a role for Membrin in synaptic function, and provides a comprehensive explanatory basis for genotype-phenotype relationships in GOSR2-PME.

Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a (59)Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect.

Background: The homozygous missense mutation c. 430G>T (p.G144W) in the GOSR2 gene has been repeatedly shown to cause progressive myoclonus epilepsy/ataxia. Thus far, no other disease associated GOSR2 mutation has been reported. Methods: From epilepsy, movement disorder and genetic clinics 43 patients suffering from progressive myoclonus epilepsy/ataxia were screened for defects in GOSR2, SCARB2 and CSTB. Results: A 61-year-old female patient suffering from progressive myoclonus epilepsy was found to be compound heterozygous for the known c.430G>T and a novel c.491_493delAGA (p.K164del) GOSR2 mutation. This is so far the oldest GOSR2 patient and her disease course seems overall milder. Conclusions: This finding further highlights the GOSR2 gene as a cause of progressive myoclonus epilepsy and expands the genotype for a potentially weaker disease allele.Progressive myoclonus epilepsies (PMEs) or ataxias are a group of neurological syndromes characterized by myoclonus, ataxia, epilepsy, and often cognitive decline, which worsen over time. 1 PMEs can clinically be subdivided depending on whether cognitive decline is a prominent feature, 1 and mode of inheritance can be a further clue toward the causative gene. Four genes are known to be associated with autosomal-recessive PME with largely preserved intellect: CSTB (MIM 601145) 2 ; SCARB2 (MIM 602257) 3,4 ; PRICKLE1 (MIM 608500) 5 ; and GOSR2 (MIM 604027). 6 Recently, a heterozygous de novo mutation in KCNC1 (MIM 176258) has been shown to cause the same phenotype. 7 The most prominent PME with largely preserved cognition is Unverricht-Lundborg disease (ULD; MIM 254800) resulting from mutations in the CSTB gene. 2 Mutations in Golgi SNAP receptor complex member 2 (GOSR2) have been shown to cause an ULD-like phenotype. 6,8,9 However, this disease usually onsets earlier than ULD, around age 2, with myoclonus and ataxia. Frequently, generalized tonic-clonic seizures then develop. Owing to rapid progression of action myoclonus and ataxia, patients are often left wheelchair bound already in their first or second decade. Thus far, all 17 reported patients with GOSR2-mediated PME have been shown to carry the same homozygous c.430G>T (p.G144W) mutation, the result of a founder effect. 6,8,9 No other GOSR2 mutation has thus far been shown to be associated with PME. MethodsWe assembled a cohort of 43 single patients or family probands showing a clinical presentation suggestive for progressive myoclonus epilepsy/ataxia syndrome. Patients were negative for mutations in ATN1, mitochondrial A8344G, and A3243G. The study was approved by the local ethical board, and informed consent was given by all patients. All 12 scavenger receptor class B, member 2 (SCARB2) exons and GOSR2 c.430G>T were screened by Sanger sequencing. ResultsWe report here on the analysis of a series of 43 PME families/ patients. Twelve patients were found to have CSTB mutations. Sequencing the remaining 31 probands found no SCARB2

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Mutations in the Golgi SNARE protein Membrin (encoded by the GOSR2 gene) cause progressive myoclonus epilepsy (PME). Membrin is a ubiquitously important protein mediating ER-to-Golgi membrane fusion, and hence it is unclear how these mutations result in a disorder restricted to the nervous system. Here we use a multi-layered strategy to elucidate the consequences of Membrin mutations from protein to neuron. We show that the pathogenic mutations cause partial reductions in SNARE-mediated membrane fusion.Importantly, these alterations were sufficient to profoundly impair dendritic growth in novel Drosophila models of GOSR2-PME. We also observed axonal trafficking abnormalities in this model, as well as synaptic malformations, trans-synaptic instability and hyperactive synaptic transmission. Our study highlights how dendritic growth is vulnerable even to subtle secretory pathway deficits, uncovers a previously uncharacterized role for Membrin in synaptic function, and provides a comprehensive explanatory basis for genotype-phenotype relationships in GOSR2-PME.