An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats in Drosophila caused adult-onset neurodegeneration attributable to poly-(glycine-arginine) proteins. Thus expanded repeats promoted neurodegeneration through neurotoxic proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence showed both poly-(glycinearginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are adult-onset, neurodegenerative diseases associated with personality change, language dysfunction and † Corresponding authors. a.isaacs@prion.ucl.ac.uk; l.partridge@ucl.ac.uk. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts progressive muscle weakness. These syndromes overlap genetically and pathologically, and can also co-occur in individuals, and within families (1). An intronic GGGGCC hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of both FTD and ALS (C9FTD/ALS) (2-4), and can be found in patients diagnosed with all common neurodegenerative diseases (5). Healthy individuals carry fewer than 33 hexanucleotide repeats, with 2 repeats being the most common, but C9FTD/ALS cases carry between 400 and 4400 repeats (2, 5, 6).The repeat expansion could cause disease by three possible mechanisms: i) toxic sense and/or antisense repeat RNA species that sequester key RNA-binding proteins, ii) toxic dipeptide repeat (DPR) proteins, generated by repeat-associated, non-ATG (RAN) translation, or iii) reduced expression of C9orf72. The absence of a severe phenotype in a homozygous C9orf72 mutation case (7), and the lack of C9orf72 coding mutations (8) argue against loss-of-function as a primary mechanism. Neuronal aggregates of RNA, termed RNA foci, generated from both sense and antisense repeat transcripts are frequent in C9FTD/ALS patient brain (9-13). The GGGGCC repeat can be translated in all sense and antisense frames, two of which encode the same DPR, resulting in five DPR proteins, all of which form inclusions in widespread brain regions (10,12,(14)(15)(16)(17)(18). It is therefore of fundamental importance to understand the contributions of repeat RNA and DPR proteins to C9orf72-mediated neurodegeneration.A major obstacle in the investigation of large expanded repeats is that they are inherently unstable. We used recombination-deficient E. coli and a cloning strategy termed recursive directional ligati...

An expanded GGGGCC repeat in a non-coding region of the C9orf72 gene is a common cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis. Non-coding repeat expansions may cause disease by reducing the expression level of the gene they reside in, by producing toxic aggregates of repeat RNA termed RNA foci, or by producing toxic proteins generated by repeat-associated non-ATG translation. We present the first definitive report of C9orf72 repeat sense and antisense RNA foci using a series of C9FTLD cases, and neurodegenerative disease and normal controls. A sensitive and specific fluorescence in situ hybridisation protocol was combined with protein immunostaining to show that both sense and antisense foci were frequent, specific to C9FTLD, and present in neurons of the frontal cortex, hippocampus and cerebellum. High-resolution imaging also allowed accurate analyses of foci number and subcellular localisation. RNA foci were most abundant in the frontal cortex, where 51 % of neurons contained foci. RNA foci also occurred in astrocytes, microglia and oligodendrocytes but to a lesser degree than in neurons. RNA foci were observed in both TDP-43- and p62-inclusion bearing neurons, but not at a greater frequency than expected by chance. RNA foci abundance in the frontal cortex showed a significant inverse correlation with age at onset of disease. These data establish that sense and antisense C9orf72 repeat RNA foci are a consistent and specific feature of C9FTLD, providing new insight into the pathogenesis of C9FTLD.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1200-z) contains supplementary material, which is available to authorized users.

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ˜ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37‐fold improvement in potency over dynasore for liposome‐stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36‐fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin‐dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin‐independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity‐dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non‐specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin‐mediated endocytosis.

Synaptic vesicles (SVs) are retrieved by more than one mode in central nerve terminals. During mild stimulation, the dominant SV retrieval pathway is classical clathrin-mediated endocytosis (CME). During elevated neuronal activity, activity-dependent bulk endocytosis (ADBE) predominates, which requires activation of the calcium-dependent protein phosphatase calcineurin. We now report that calcineurin dephosphorylates dynamin I in nerve terminals only above the same activity threshold that triggers ADBE. ADBE was arrested when the two major phospho-sites on dynamin I were perturbed, suggesting that dynamin I dephosphorylation is a key step in its activation. Dynamin I dephosphorylation stimulates a specific dynamin I-syndapin I interaction. Inhibition of this interaction by competitive peptides or by site-directed mutagenesis exclusively inhibited ADBE but did not affect CME. The results reveal that the phospho-dependent dynamin-syndapin interaction recruits ADBE to massively increase SV endocytosis under conditions of elevated neuronal activity.

Bulk endocytosis in central nerve terminals is activated by strong stimulation; however, the speed at which it is initiated and for how long it persists is still a matter of debate. To resolve this issue, we performed a characterization of bulk endocytic retrieval using action potential trains of increasing intensity. Bulk endocytosis was monitored by the loading of the fluorescent dyes FM2-10 and FM1-43, uptake of tetramethylrhodamine-dextran (40 kDa), or morphological analysis of uptake of the fluid-phase marker horseradish peroxidase. When neuronal cultures were subjected to mild stimulation (200 action potentials at 10 Hz), bulk endocytosis was not observed using any of our assay systems. However, when more intense trains of action potentials (400 or 800 action potentials at 40 and 80 Hz, respectively) were applied to neurons, bulk endocytosis was activated immediately, with the majority of bulk endocytosis complete by the end of stimulation. This contrasts with single synaptic vesicle endocytosis, the majority of which occurred after stimulation was terminated. Thus, bulk endocytosis is a fast event that is triggered during strong stimulation and provides the nerve terminal with an appropriate mechanism to meet the demands of synaptic vesicle retrieval during periods of intense synaptic vesicle exocytosis.