6, p < 0 001)—i e , with temporal proximity from the motor respon

6, p < 0.001)—i.e., with temporal proximity from the motor response. This is to be expected from a response preparation signal driven by large temporal fluctuations in sensory

input ( Yang and Shadlen, 2007). We carried out additional analyses locked to the onset of the response period, which all confirmed that motor beta-band activity behaved as a response preparation signal (Figure S7): (1) the neural encoding of the sum of response updates distinguished correct choices from errors from more than 500 ms before the onset of the response period KU-55933 price (paired t test, t14 = 4.8, p < 0.001); (2) the neural decoding of choice (i.e., left- versus right-handed response) showed similar predictive profiles preceding correct choices and errors (see Supplemental Information); and (3) the between-element variability in neural encoding of response updates

correlated positively with Selleck CP690550 the between-element weighting profile estimated behaviorally (r = +0.44 ± 0.10, t test against zero, t14 = 4.4, p < 0.001). Finally, we assessed whether the neural encoding of DUk in motor beta-band activity also fluctuated rhythmically according to the phase of parietal delta oscillations (Figure 7C), and found that it followed the same phase relationship as its earlier encoding in broadband parietal signals (Rayleigh test, r14 = 0.50, p < 0.01). This phase dependency suggests that motor beta-band activity reflects a computation that occurs downstream from the weighting of momentary evidence according

to the phase of parietal delta oscillations. Together, these findings chart the electrophysiological substrates of the sensorimotor cascade whereby successive samples 3-mercaptopyruvate sulfurtransferase of sensory evidence are processed from lower to higher levels, integrated, and converted into an appropriate response. By linking trial-to-trial fluctuations in neural signals to variability in choice, these findings draw a clear distinction between the computations performed by two neural mechanisms during categorical decision making. First, momentary evidence undergoes a multiplicative weighting according to the phase of ongoing delta oscillations (1–3 Hz) overlying human parietal cortex. Subsequently, lateralized beta-band activity (10–30 Hz) over the motor cortex integrates the weighted evidence in an additive fashion, consistent with the formation of a decision variable. Categorical choices are thus preceded by discrete central and motor stages, both of which follow an early perceptual stage confined to early visual cortex. These findings thus call into question the widely held view that evidence accumulation is indistinguishable from the gradual engagement of a response effector—in other words, that the neural encoding of decision-relevant evidence reduces to a preparatory signal that precedes motor output ( Shadlen and Newsome, 2001; Roitman and Shadlen, 2002; Gold and Shadlen, 2003, 2007).

How exactly IFG is involved in the evaluative process itself is s

How exactly IFG is involved in the evaluative process itself is still unclear. Our results suggest that, if it has a causal role in promoting nondefault riskier choices, then its disruption would lead to taking safer, default choices. In agreement

with the possibility that IFG or an adjacent lateral frontal region is involved in dynamic, context-dependent changes in decision making, one recent study applied transcranial magnetic stimulation in this vicinity and found that subjects were more likely to make socially unbiased decisions and to integrate considerations of reward magnitudes in the standard manner (Baumgartner et al., 2011), rather than taking the social context into Selleckchem Apoptosis Compound Library consideration. Eighteen subjects (nine women and nine men), aged 22–36 years, completed the task. They were paid £10 plus a performance-dependent

bonus of between £15 and £30. Ethical approval was given by the Oxfordshire National Health Service Research Ethics Committee (local ethics code: 07/Q1603/11). Before fMRI scanning, every subject selleck screening library was instructed in the task and played a shorter version of the task used in the fMRI experiment for about 10 min. The behavioral task in the scanner consisted of 24 blocks, each composed of eight trials (192 decisions in total) in which the subjects had to decide between a safer option with a higher reward probability but a lower reward magnitude and a riskier option with a potentially higher reward magnitude but lower reward probability. MYO10 There were eight decisions, and they were each presented once in each block in a randomized order that varied. In this way, we were conclusively able to show that dynamic changes in decisions occur, because of sensitivity to risk pressure, even when the exact same options were presented. Risk pressure varied because all eight decisions were associated with different values and were presented in different orders, with different outcomes, and in the context of different block target values (which the subjects had to reach in order to keep the points they won during the

block). Four target levels were used in the experiments. The different target levels helped ensure that risk pressure (see Introduction and the following section) had some parametric range. To equalize expected gains at the beginning of a block regardless of target level and to keep motivation relatively stable, we introduced a “multiplier,” which was displayed on top of the “target” line. The multiplier indicated a factor that would be used to multiply the points subjects won before they were added to the subject’s account if they reached the target. We chose the multiplication factor by applying our model (discussed in the next section) to generate equal expected gains at the first trial of a block. Simply put, if a participant had a high target to reach, all his points were multiplied (e.g., by 2) if he managed to reach it.

In fact, in the present study the morphological characteristics d

In fact, in the present study the morphological characteristics described for selleck products different macrophage differentiation times indicated the presence of granulocytes was very low (<5%). Additionally, the results related to morphologic analysis, phagocytosis, microbicidal

activity, enzymatic NAG and MPO activity, and the previous reports in the literature confirm that the ideal culture condition of canine monocyte differentiation into macrophages is obtained after 5 days of in vitro monocyte incubation. The canine immune system has several peculiarities, especially in relation to the number of circulating granulocytes in the blood stream. Neutrophils present high expression of the CD4+ molecule (Williams, 1997), and this feature interferes with the purification of CD4+ T cells with high purity using typical methods of separation. Thus, using peripheral blood samples and performing CD4+ T-cell separation, increased contamination by canine neutrophils cannot be avoided. The best alternative for establishing a purification system was to carry it out on the fifth day of monocyte differentiation, when lower levels of granulocytes are present. This strategy allowed an increased performance of CD4+ or CD8+ purity level (≥90%) using

magnetic KU-55933 concentration column methodology (Fig. 6). The data presented here describe the ideal conditions for in vitro differentiation of monocytes, derived from canine peripheral blood, into macrophages. Based on our data presented here, we concluded that monocytes differentiate into macrophages over the course of 5 days and displayed an intermediate frequency of parasitism and parasite load 72 h after L. chagasi infection. At this time, the inclusion of purified CD4 and/or CD8 T cells in infected macrophages culture would be useful for analyzing the impact of modulation in in vitro parasitism. Furthermore, the purification system using canine T-lymphocyte subsets after 5 days Florfenicol of monocyte differentiation

proved to be efficient for obtaining cultures permitting high CD4 or CD8 T-cell purity (≥90%). Thus, the use of co-culture systems employing canine monocytes differentiated into macrophages and purified CD4+ and/or CD8+ T cells may contribute to the analysis of the adaptive immune response in dogs. This methodology could be incorporated in vaccine and treatment studies against CVL that aim to analyze the microbicidal potential induced by specific CD4+ and/or CD8+ T cells. The authors are grateful for the use of the facilities at CEBIO, Universidade Federal de Minas Gerais and Rede Mineira de Bioterismo (FAPEMIG). This work was supported by Fundação de Amparo a Pesquisa do Estado de Minas Gerais, Brazil (grant: CBB-APQ-02473-10; CBB-APQ-00356-10-PPSUS; CBB-APQ-01052-11; APQ-01698-12), Conselho Nacional de Desenvolvimento Científico e Tecnológico- CNPq, Brazil (grant: 403485/2008-8 – PAPES V/FIOCRUZ; 473234/2010-6; 560943/2010-5; 310129/2011-7; 482249/2012-9) and CAPES.

At this point we asked whether the information on the value of th

At this point we asked whether the information on the value of the odor conveyed by the synchronized firing trains diverged between the two odors at a time in the trial before the

animal made a decision. We performed principal component (PC) analysis of divergent synchronized pair responses to the odors. Figure S3A shows, for the first and best blocks, the time course for the responses to odors PD0332991 research buy in 2D PC space, and Figure S3B shows the time course of the Euclidean distance in PC space between the points for the rewarded and unrewarded odors. There is clear divergence of the responses to the odors in the best block, but not in the first block. Figure 4C shows the p value for a ranksum test of divergence of the Euclidean distance between rewarded and unrewarded odors. Divergence of synchronized unit firing becomes significant at ∼1 s (0.7 s after addition of the odor), which is ∼0.25 s earlier than the time at which the animals make a decision to stop licking to the unrewarded odor (1.25 s, estimated with a ranksum test on licks). A fraction of a second afterward at ∼1.7 s, the mice change their sniff frequency (Figure 1Bii). BIBF 1120 mw Thus, the divergence between rewarded and unrewarded odors for synchronized trains carries

information that the animal can use for odor discrimination. We next asked whether analysis of trials where the animals made mistakes shows that synchrony reflected responses to odor, and not responses that mirrored the behavioral action. In other words, when the animal makes a mistake and licks on the water tube to obtain a reward why when exposed to the unrewarded odor (false alarm), are the synchronized spike trains more like the synchronized firing that takes place when the animal

correctly licks for a water reward to a rewarded odor (hit), or more like the synchronized responses when the animal correctly does not lick for the unrewarded odor (correct rejection)? As shown by the z-score cumulative histograms in Figure 5, the synchronized spiking decreased (Δz < 0) in response to the unrewarded odor, regardless of whether the animal licked during this odor (false alarm, green) or not (correct rejection, black). Similarly, for the majority of the trials, synchronized firing increased (Δz > 0) in response to the rewarded odor whether the animal licked during this odor (hit, blue) or refrained from licking (miss, red). Thus, the odor-induced changes in synchronized firing are responses to the odor as opposed to responses that follow the animal’s behavior or licking. In addition, because the responses follow the odor presented rather than the movement the animal made, the data in this figure indicate that the synchronized spike trains are not brought about by noise caused by the animal’s movements. The percent of unit pairs whose synchronized spike trains respond differentially to the odors decreased as a function of distance between electrodes (Figure 6A, blue).

, 2006) or the transfection of cells in a culture dish with const

, 2006) or the transfection of cells in a culture dish with constructs that limit synaptic vesicle release and hence leave postsynaptic targets that receive both chronically “silenced” and normal terminals (Béïque et al., 2011, Harms and Craig,

2005, Hou et al., 2008 and Lee et al., 2010). The results of studies on ssHSP have been varied, but some groups indicate a compensatory increase in the expression of AMPARs exclusively at the chronically silenced synapses and not at nearest normal neighbor synapses (Lee click here et al., 2010, Hou et al., 2008 and Béïque et al., 2011). Until now, no group has managed the difficult technical feat of persistently activating single synapses among normal neighbors on a given neuron. Action potential firing and synaptic vesicle release from a single presynaptic neuron can be induced by current injection after whole-cell configuration has been achieved in patch-clamp electrophysiology. However, achieving stable firing for a prolonged period in the activated neuron and determining the synapses coming from the activated cell onto a receiving cell are technically difficult. An alternative strategy is therefore needed to elicit sustained yet selective presynaptic activity. In order to persistently activate some of the axon terminals in a neuronal culture, Hou

and colleagues transfected light-activated glutamate receptor 6 (LiGluR) subunits sparsely into cultured cortical neurons. LiGluR Sirolimus subunits form a normal cation-permeant channel, which is activated only when UV light (380 nm) photoconverts the tethered agonist MAG and is inactivated Ketanserin when blue light (480 nm) catalyzes the reverse isomerization (Szobota et al., 2007). Thus, LiGluR enables light-controlled depolarization, action potential firing, Ca2+ rises, and consequent glutamate release from axonal terminals just in activated neurons.

Due to the low transfection efficiency in the system created by Hou and colleagues, only a few neurons in each dish expressed LiGluR. This ensured that some cells received synaptic input from both light-controlled terminals from LiGluR-expressing neurons and normal terminals from non-LiGluR-expressing neurons. To distinguish the light-controlled terminals from the normal terminals, the authors introduced yellow fluorescent protein-labeled synapsin1 (syn-YFP) to the cells expressing LiGluR (Figure 1). This approach was designed to enable comparison between persistently activated synapses and normal neighbors on the same postsynaptic cell. The method of Hou and colleagues contrasts with a recent study using a different light-activated channel that showed that persistently exciting a single neuron of interest leads to homeostatic postsynaptic changes on that same neuron (Goold and Nicoll, 2010).

Sequence analyses revealed that this gene is a relic of the B-typ

Sequence analyses revealed that this gene is a relic of the B-type Pcdhg isoforms, and its promoter region also contains a conserved sequence element (CSE) found in most Pcdh genes ( Figures S4E–S4G). The expression levels of most Pcdhg isoforms are not affected by the deletion of C-type genes except for a few neighboring ones which are upregulated, and buy Quisinostat quantification of constant exon reads indicated that the combinatorial expression levels of the remaining Pcdhg genes in Pcdhgtcko/tcko mice are ∼75% of the wild-type levels ( Figures S4C and S4D and Table S1). Thus, the loss

of function of the C-type isoforms cannot be compensated by other Pcdhg isoforms. Many Pcdhb genes (as well as AK149307) are marginally upregulated in the this website Pcdhgtcko/tcko mice, likely also due to the action of the Pcdhb cluster enhancer as mentioned above. In addition, no neomorphic Pcdhg variants were detected in Pcdhgtcko/tcko mutants with splice junction analysis of the RNA-Seq data ( Table S2). The striking phenotypic similarities in contrast to the vastly distinct Pcdh repertoires in Pcdhgtcko/tcko and Pcdhgdel/del mutants suggest that lack of the C-type Pcdhg isoforms themselves, which is common for both mutants, is the primary cause of the common phenotypes. Since the primary phenotype observed

in both Pcdhgtcko/tcko and Pcdhgdel/del is neuronal cell death, we crossed both mutant lines to Bax knockout mice ( Knudson et al., 1995) to compare phenotypes when neuronal apoptosis

is genetically blocked. Consistent Urease with previous observations ( Weiner et al., 2005), Pcdhgdel/del;Bax−/− pups show improved neurological function as compared with Pcdhgdel/del mutants, yet they still lack voluntary movements, and despite considerable efforts we were unable to recover any Pcdhgdel/del;Bax−/− mutants beyond P0 ( Table 1 and Movie S2). Surprisingly, however, while some Pcdhgtcko/tcko;Bax−/− mutants die at P0, many live substantially longer despite being weaker and smaller than wild-type and heterozygous pups. By culling littermates we were able to recover a number of Pcdhgtcko/tcko;Bax−/− mutants at weaning age. Some of these animals survived up to 6 months, although their persistent ataxia indicates neurological impairment ( Table 1 and Movie S2). As described for the Pcdhgdel/del;Bax−/− mutants ( Prasad et al., 2008; Weiner et al., 2005), the morphology of spinal cord sections of Pcdhgtcko/tcko;Bax−/− is indistinguishable from that of the Pcdhg+/+;Bax−/− animals, showing no signs of astrogliosis or microglia activation, and the arborization patterns of IaPA terminals appear largely indistinguishable from those of the controls ( Figure S5A). Counts of both VGAT+ and VGLUT1+ inputs onto motor neurons were normal in the Bax−/− genetic background, while VGLUT2+ and VAChT+ synapses remain unchanged ( Figure S5B).

, 2005) The firing fields formed a grid-like pattern, and the ce

, 2005). The firing fields formed a grid-like pattern, and the cells were referred to as grid cells (Figure 1). The size of each grid field and the spacing between them were learn more found to increase progressively from small in dorsal to large in ventral MEC (Fyhn et al., 2004, Hafting et al., 2005 and Sargolini et al., 2006). At the dorsal tip, the spacing was approximately 30 cm in the rat; at the ventral tip, it was more

than 3 m (Brun et al., 2008). The position of the grid vertices in the x,y plane (their grid phase) appeared to vary randomly between cells at all dorsoventral locations, but each grid maintained a stable grid phase over time. The cells fired at the same x,y positions irrespective of changes in the animal’s speed and direction, and the firing fields persisted in darkness, suggesting that self-motion

information is used actively by grid cells to keep track of the animal’s position in the environment (Hafting et al., 2005 and McNaughton et al., 2006). This process, referred to as path integration, may provide the metric Roxadustat ic50 component of the spatial map. Grid cells were soon found to colocalize with several other specialized cell types. A substantial portion of the principal cells in layer III and layers V and VI of the MEC were tuned to direction, firing if and only if the animal’s head faced a certain angle relative to its immediate surroundings (Sargolini et al., 2006). Similar cells were already known to exist in other parahippocampal and subcortical regions (Ranck, 1985 and Taube, 2007), but the entorhinal head direction cells were different in that many of them exhibited grid-like activity at the same time (conjunctive grid × head direction cells). In addition, approximately 10% of the active entorhinal cell population was found to fire selectively in the vicinity of geometric borders such

as the walls of a recording enclosure or the edges of a table (Savelli et al., 2008 and Solstad et al., 2008). We have referred to these cells as border cells (Solstad TCL et al., 2008). Collectively, grid cells, head direction cells, and border cells are thought to form the neural basis of a metric representation of allocentric space (Moser et al., 2008). The entorhinal spatial representation is different from the hippocampal map in that cell assemblies maintain their intrinsic firing structure across environments. If two grid cells have similar vertices in one environment, they will fire at similar locations also in another environment (Fyhn et al., 2007 and Hafting et al., 2005). If two border cells fire along adjacent borders in one enclosure, they will do so in other boxes, too (Solstad et al., 2008). In the hippocampus, in contrast, different subsets of neurons are recruited in different environments (Muller et al.

To investigate the interaction of PICK1

with Arf1, we per

To investigate the interaction of PICK1

with Arf1, we performed GST-PICK1 pull-down assays with a constitutively active mutant of Arf1 (Arf1Q71L) or a nucleotide-binding-defective mutant (Arf1T31N) expressed in COS cells. GST-PICK1 interacts specifically with the constitutively INCB024360 mouse active Arf1Q71L mutant, showing negligible binding to Arf1T31N, suggesting that the PICK1-Arf1 interaction is GTP dependent (Figure 1A). To test this further, we carried out GST-PICK1 pull-down assays with purified his6-Arf1 in the absence of other proteins and in the presence of either nonhydrolyzable GTP (GTPγS) or guanosine diphosphate (GDPβS). Arf1 binds PICK1 only in the presence of GTPγS, demonstrating a direct GTP-dependent interaction of Arf1 with PICK1 (Figure 1B). To investigate the PICK1-Arf1 interaction in native tissue, we carried out coimmunoprecipitations (co-IPs) from neuronal extracts using PICK1 antibodies in the presence or absence of GTPγS.

Arf1 interacts with PICK1 only in the presence of GTPγS, demonstrating that a GTP-dependent PICK1-Arf1 complex exists in neurons (Figure 1C). The GluA2-PICK1 interaction is unaffected by the presence of GTPγS (Figure 1C and Figure S1A available online). Since a major function of PICK1 is regulating AMPAR trafficking via an interaction with the GluA2 subunit, we assessed whether PICK1 can complex with both GluA2 and Arf1 simultaneously. Co-IP from cultured neuronal extracts using anti-GluA2 antibodies demonstrates that Arf1 is in a

GTP-dependent complex with GluA2 (Figure 1D). The Apoptosis Compound Library GluA2-Arf1 complex is disrupted following transduction of neurons with Sindbis virus expressing a peptide (pep2-EVKI) that inhibits AMPAR-PICK1 interactions (Terashima et al., 2004 and Terashima et al., 2008), demonstrating that Arf1 associates with GluA2 via PICK1 (Figures 1D and S1B). To confirm AMPAR subunit specificity of this interaction, we carried out co-IP experiments from transfected HEK293 all cells. Endogenous Arf1 forms a complex with PICK1flag and mycGluA2 but not mycGluA1 (Figure S1C). To analyze the subcellular distribution of Arf1, we carried out differential detergent fractionation of synaptosomes prepared from brain tissue. It has previously been shown that PICK1 is present in synaptosomal fractions as well as PSD fractions I and II but not the core PSD III fraction (Rocca et al., 2008 and Xia et al., 1999). Arf1 shows a strikingly similar distribution to PICK1, demonstrating that both proteins are found in the same subcellular fractions, and are both loosely associated with the postsynaptic density (Figure 1E). Arf6 has also been implicated in AMPAR trafficking during LTD (Scholz et al., 2010), so we investigated whether this related protein binds PICK1. GST pull-downs demonstrate that Arf6 does not interact with PICK1 (Figure 1F).

The F2 progeny were scored to identify animals that lacked GFP ex

The F2 progeny were scored to identify animals that lacked GFP expression under normoxic conditions. To map n5500, the polymorphic Hawaiian CB4856 strain was crossed with n5500; nIs470 animals to obtain F2 progeny for SNP mapping ( Davis et al., 2005). To map the n5500 suppressor n5515 using genetic markers, n5500 II; nIs470 IV; n5515 males were crossed with n5500 II; nIs470 IV; dpy-6(e14) egl-15(n484) X hermaphrodites. Seven out of fifteen Compound Library ic50 Egl non-Dpy F2 progeny segregated GFP-negative n5500-suppressed animals. Refined mapping using SNP analysis further positioned n5515 between dpy-6 and egl-15 in an interval between the SNPs pk6127 and pk6138. To map the dominant suppressor

n5535, n5500; nIs470; n5535 hermaphrodites were crossed with n5500; nIs470 males, and GFP-positive F2 animals were isolated for SNP mapping. Locomotive responses to step changes of O2 were measured using a custom-built multiworm tracker and a gas-flow system controlled in real-time by MATLAB (see Figure S1). The gas flow consisted of pre-mixed 20%, 10%, selleck compound 5%, or 0% O2 balanced by N2. Well-fed young adult hermaphrodites (50–100 per assay) were transferred to a Petri plate freshly seeded with the bacterium OP50 and allowed to stabilize for 1 hr before the assay at 20°C. Worm-tracking

videos were analyzed later using MATLAB to calculate instantaneous locomotion speeds and other behavioral parameters. A hypoxia chamber (Coy Laboratory) that contained 0.5% O2 balanced by N2 was used for experiments involving hypoxia experience. After 24 hr of hypoxia exposure, animals

were allowed to recover in room air at 20°C for 2 hr preceding the acute behavioral assay. For experiments involving H2S exposure, 1 μl of 0.1M NaHS, an established H2S donor that releases 3-mercaptopyruvate sulfurtransferase H2S from solution, was dropped on the edge of agar-containing Petri plates and immediately sealed with tape to ensure airtight conditions. To obtain optimal effects, 24 hr duration of H2S exposure was used for behavioral experiments; 12 hr duration was used for GFP induction; and 1 hr duration was used for biochemical interaction experiments and quantitative real-time PCR. We thank Jo Anne Powell-Coffman, Yuichi Iino, Rene Garcia, Michael Hengartner, Mark Roth, Andy Fire, and Erik Jorgensen for reagents; the Caenorhabditis Genetics Center for strains; Na An, Rita Droste, and Tove Ljungars for technical assistance; Ales Hnizda, Jakub Krijt, and Milan Kodicek for help with characterization of purified CYSL-1; Viktor Kozich for helpful discussion; and Shunji Nakano, Takashi Hirose, Nick Paquin, Howard Chang, and Shuo Luo for comments. This work was supported by grant GM24663 to H.R.H from the NIH. R.V. was supported by grant No. 21709 from Grant Agency of Charles University, Prague, Czech Republic and by the Research Project of Charles University No. MSM0021620806. H.R.H.

, 2000) The human and iPSC data in the current study suggest tha

, 2000). The human and iPSC data in the current study suggest that ADARB2 could play a role in human C9ORF72 ALS by interacting with the C9ORF72 RNA foci. One hypothesis Protease Inhibitor Library is that the interaction of this RBP with the GGGGCCexp RNA might result in its loss of function. Additional studies to investigate the downstream effects of the loss of ADARB2 on biological processes such as RNA editing would be important to further characterize the roles of this protein in CNS cytotoxicity. While we have only thus far investigated

one RBP interactor identified in the present screen (Table S4), further studies are required to determine whether any of the other RBPs might interact with the GGGGCCexp RNA in vivo. Moreover, a GC-rich scrambled RNA sequence was used, which probably forms a G-quadruplex similar to the GGGGCC × 6.5 RNA (Fratta et al., 2012 and Reddy et al., 2013), thereby enriching for structure and sequence-specific protein interactors. However, the stringency of this analysis might have inadvertently excluded RBPs whose binding to the GGGGCC RNA was not apparent since it also showed an affinity to the GC rich scrambled

RNA structure. Finally, it is possible that multiple RBPs UMI-77 cost bind to the expansion, leading to either additive or synergistic effects, implicating various downstream pathways. RAN protein has been reported in C9ORF72 tissue and hypothesized to contribute to disease toxicity. Our studies document that C9ORF72 iPSC neurons have at least

one RAN protein and thus recapitulate the pathology seen in human C9ORF72 postmortem brain. However, despite the mitigation of excitotoxicity, large reductions in RNA foci and RBP aggregation and the normalization of various genes by the ASO therapy, RAN peptide can still be detected after ASO treatment. This would suggest that the detected accumulated cytoplasmic peptide was not a major contributor to neurotoxicity in our culture model. Although it is important to note that it is possible that our ASO treatment could rescue many the formation of newly synthesized RAN peptides and that longer ASO treatment would eventually show a reduction of RAN products. Furthermore, there could be other RAN peptides such as, e.g., antisense RAN peptides, contributing to the observed phenotypes, but that were not detected with the present antibodies, since our antibody preferentially detects the poly-(Gly-Pro) RAN product. While C9ORF72 protein exhibits structural similarity to the DENN protein family, the function of the C9ORF72 protein remains unclear (Levine et al., 2013). DENN proteins are GTP-GDP exchange factors (GEFs) for Rab GTPases, thus implicating the C9ORF72 protein in vesicular trafficking (Zhang et al., 2012). C9ORF72 RNA levels are reduced in patients that contain the expanded allele and a recent study has suggested that C9ORF72 haploinsufficiency causes neurodegeneration in a zebrafish model (Ciura et al.