Ketogenic diet and other dietary treatments for epilepsy.

BACKGROUND: The ketogenic diet (KD), being high in fat and low in carbohydrates, has been suggested to reduce seizure frequency. It is currently used mainly for children who continue to have seizures despite treatment with antiepileptic drugs. Recently, there has been interest in less restrictive KDs including the modified Atkins diet (MAD) and the use of these diets has extended into adult practice. OBJECTIVES: To review the evidence for efficacy and tolerability from randomised controlled trials regarding the effects of KD and similar diets. SEARCH METHODS: We searched the Cochrane Epilepsy Group's Specialized Register (30 March 2015), the Cochrane Central Register of Controlled Trials (CENTRAL) via the Cochrane Register of Studies Online (CRSO, 30 March 2015), MEDLINE (Ovid, 30 March 2015), ClinicalTrials.gov (30 March 2015) and the WHO International Clinical Trials Registry Platform (ICTRP, 30 March 2015). We imposed no language restrictions. We checked the reference lists of retrieved studies for additional reports of relevant studies. SELECTION CRITERIA: Studies of KDs and similar diets for people with epilepsy. DATA COLLECTION AND ANALYSIS: Two review authors independently applied pre-defined criteria to extract data and assessed study quality. MAIN RESULTS: We identified seven randomised controlled trials that generated eight publications.All trials applied an intention-to-treat analysis with varied randomisation methods. The seven studies recruited 427 children and adolescents and no adults. We could not conduct a meta-analysis due to the heterogeneity of the studies.Reported rates of seizure freedom reached as high as 55% in a 4 : 1 KD group after three months and reported rates of seizure reduction reached as high as 85% in a 4 : 1 KD group after three months.One trial found no significant difference between the fasting-onset and gradual-onset KD for rates of seizure freedom and reported a greater rate of seizure reduction in the gradual-onset KD group.Studies assessing the efficacy of the MAD reported seizure freedom rates of up to 10% and seizure reduction rates of up to 60%. One study compared the MAD to a 4 : 1 KD, but did not report rates of seizure freedom or seizure reduction.Adverse effects were fairly consistent across different dietary interventions. The most commonly reported adverse effects were gastrointestinal syndromes. It was common that adverse effects were the reason for participants dropping out of trials. Other reasons for drop-out included lack of efficacy and non-acceptance of the diet.Although there was some evidence for greater antiepileptic efficacy for a 4 : 1 KD over lower ratios, the 4 : 1 KD was consistently associated with more adverse effects.No studies assessed the effect of dietary interventions on quality of life, or cognitive or behavioural functioning. AUTHORS' CONCLUSIONS: The randomised controlled trials discussed in this review show promising results for the use of KDs in epilepsy. However, the limited number of studies, small sample sizes and a sole paediatric population resulted in a poor overall quality of evidence.There were adverse effects within all of the studies and for all KD variations, such as short-term gastrointestinal-related disturbances, to longer-term cardiovascular complications. Attrition rates remained a problem with all KDs and across all studies, reasons for this being lack of observed efficacy and dietary tolerance.There was a lack of evidence to support the clinical use of KD in adults with epilepsy, therefore, further research would be of benefit.Other more palatable but related diets, such as the MAD ketogenic diet, may have a similar effect on seizure control as classical KD but this assumption requires more investigation. For people who have medically intractable epilepsy or people who are not suitable for surgical intervention, a KD remains a valid option; however, further research is required.

CREB phosphorylation at Ser133 regulates transcription via distinct mechanisms downstream of cAMP and MAPK signalling.

CREB (cAMP-response-element-binding protein) is an important transcription factor for the activation of a number of immediate early genes. CREB is phosphorylated on Ser133 by PKA (protein kinase A), promoting the recruitment of the co-activator proteins CBP (CREB-binding protein) and p300; this has been proposed to increase the transcription of CREB-dependent genes. CREB is also phosphorylated on Ser133 by MSK1/2 (mitogen- and stress-activated kinase 1/2) in cells in response to the activation of MAPK (mitogen-activated protein kinase) signalling; however, the relevance of this to gene transcription has been controversial. To resolve this problem, we created a mouse with a Ser133 to alanine residue mutation in the endogenous Creb gene. Unlike the total CREB knockout, which is perinatally lethal, these mice were viable, but born at less than the expected Mendelian frequency on a C57Bl/6 background. Using embryonic fibroblasts from the S133A-knockin mice we show in the present study that Ser133 phosphorylation downstream of PKA is required for CBP/p300 recruitment. The requirement of Ser133 phosphorylation for the PKA-mediated induction of CREB-dependent genes was, however, promoter-specific. Furthermore, we show that in cells the phosphorylation of CREB on Ser133 by MSKs does not promote strong recruitment of CBP or p300. Despite this, MSK-mediated CREB phosphorylation is critical for the induction of CREB-dependent genes downstream of MAPK signalling.

Munc18-1 protein molecules move between membrane molecular depots distinct from vesicle docking sites.

Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.

MSK1 regulates homeostatic and experience-dependent synaptic plasticity.

The ability of neurons to modulate synaptic strength underpins synaptic plasticity, learning and memory, and adaptation to sensory experience. Despite the importance of synaptic adaptation in directing, reinforcing, and revising the behavioral response to environmental influences, the cellular and molecular mechanisms underlying synaptic adaptation are far from clear. Brain-derived neurotrophic factor (BDNF) is a prime initiator of structural and functional synaptic adaptation. However, the signaling cascade activated by BDNF to initiate these adaptive changes has not been elucidated. We have previously shown that BDNF activates mitogen- and stress-activated kinase 1 (MSK1), which regulates gene transcription via the phosphorylation of both CREB and histone H3. Using mice with a kinase-dead knock-in mutation of MSK1, we now show that MSK1 is necessary for the upregulation of synaptic strength in response to environmental enrichment in vivo. Furthermore, neurons from MSK1 kinase-dead mice failed to show scaling of synaptic transmission in response to activity deprivation in vitro, a deficit that could be rescued by reintroduction of wild-type MSK1. We also show that MSK1 forms part of a BDNF- and MAPK-dependent signaling cascade required for homeostatic synaptic scaling, which likely resides in the ability of MSK1 to regulate cell surface GluA1 expression via the induction of Arc/Arg3.1. These results demonstrate that MSK1 is an integral part of a signaling pathway that underlies the adaptive response to synaptic and environmental experience. MSK1 may thus act as a key homeostat in the activity- and experience-dependent regulation of synaptic strength.

Staggered overdose pattern and delay to hospital presentation are associated with adverse outcomes following paracetamol-induced hepatotoxicity.

AIMS: Paracetamol (acetaminophen) poisoning remains the major cause of severe acute hepatotoxicity in the UK. In this large single centre cohort study we examined the clinical impact of staggered overdoses and delayed presentation following paracetamol overdose. RESULTS: Between 1992 and 2008, 663 patients were admitted with paracetamol-induced severe liver injury, of whom 161 (24.3%) had taken a staggered overdose. Staggered overdose patients were significantly older and more likely to abuse alcohol than single time point overdose patients. Relief of pain (58.2%) was the commonest rationale for repeated supratherapeutic ingestion. Despite lower total ingested paracetamol doses and lower admission serum alanine aminotransferase concentrations, staggered overdose patients were more likely to be encephalopathic on admission, require renal replacement therapy or mechanical ventilation and had higher mortality rates compared with single time point overdoses (37.3% vs. 27.8%, P= 0.025), although this overdose pattern did not independently predict death. The King's College poor prognostic criteria had reduced sensitivity (77.6, 95% CI 70.8, 81.5) for this pattern of overdose. Of the 396/450 (88.0%) single time point overdoses in whom accurate timings could be obtained, 178 (44.9%) presented to medical services >24 h following overdose. Delayed presentation beyond 24 h post overdose was independently associated with death/liver transplantation (OR 2.25, 95% CI 1.23, 4.12, P= 0.009). CONCLUSIONS: Both delayed presentation and staggered overdose pattern are associated with adverse outcomes following paracetamol overdose. These patients are at increased risk of developing multi-organ failure and should be considered for early transfer to specialist liver centres.

Overdose pattern and outcome in paracetamol-induced acute severe hepatotoxicity.

AIMS: Paracetamol (acetaminophen) hepatotoxicity is the commonest cause of acute liver failure (ALF) in the UK. Conflicting data regarding the outcomes of paracetamol-induced ALF resulting from different overdose patterns are reported. METHODS: Using prospectively defined criteria, we have analysed the impact of overdose pattern upon outcome in a cohort of 938 acute severe liver injury patients admitted to the Scottish Liver Transplantation Unit. RESULTS: Between 1992 and 2008, 663 patients were admitted with paracetamol-induced acute severe liver injury. Of these patients, 500 (75.4%) had taken an intentional paracetamol overdose, whilst 110 (16.6%) had taken an unintentional overdose. No clear overdose pattern could be determined in 53 (8.0%). Unintentional overdose patients were significantly older, more likely to abuse alcohol, and more commonly overdosed on compound narcotic/paracetamol analgesics compared with intentional overdose patients. Unintentional overdoses had significantly lower admission paracetamol and alanine aminotransferase concentrations compared with intentional overdoses. However, unintentional overdoses had greater organ dysfunction at admission, and subsequently higher mortality (unintentional 42/110 (38.2%), intentional 128/500 (25.6%), P < 0.001). The King's College poor prognostic criteria had reduced sensitivity in unintentional overdoses (77.8%, 95% confidence intervals (CI) 62.9, 88.8) compared with intentional overdoses (89.9%, 95% CI 83.4, 94.5). Unintentional overdose was independently predictive of death or liver transplantation on multivariate analysis (odds ratio 1.91 (95% CI 1.07, 3.43), P = 0.032). CONCLUSIONS: Unintentional paracetamol overdose is associated with increased mortality compared with intentional paracetamol overdose, despite lower admission paracetamol concentrations. Alternative prognostic criteria may be required for unintentional paracetamol overdoses.

Regulation of the miR-212/132 locus by MSK1 and CREB in response to neurotrophins.

Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.

A Role for the Autophagic Receptor, SQSTM1/p62, in Trafficking NF-κB/RelA to Nucleolar Aggresomes.

Elevated NF-κB activity is a contributory factor in many hematologic and solid malignancies. Nucleolar sequestration of NF-κB/RelA represses this elevated activity and mediates apoptosis of cancer cells. Here, we set out to understand the mechanisms that control the nuclear/nucleolar distribution of RelA and other regulatory proteins, so that agents can be developed that specifically target these proteins to the organelle. We demonstrate that RelA accumulates in intranucleolar aggresomes in response to specific stresses. We also demonstrate that the autophagy receptor, SQSTM1/p62, accumulates alongside RelA in these nucleolar aggresomes. This accumulation is not a consequence of inhibited autophagy. Indeed, our data suggest nucleolar and autophagosomal accumulation of p62 are in active competition. We identify a conserved motif at the N-terminus of p62 that is essential for nucleoplasmic-to-nucleolar transport of the protein. Furthermore, using a dominant-negative mutant deleted for this nucleolar localization signal (NoLS), we demonstrate a role for p62 in trafficking RelA and other aggresome-related proteins to nucleoli, to induce apoptosis. Together, these data identify a novel role for p62 in trafficking nuclear proteins to nucleolar aggresomes under conditions of cell stress, thus maintaining cellular homeostasis. They also provide invaluable information on the mechanisms that regulate the nuclear/nucleolar distribution of RelA that could be exploited for therapeutic purpose. IMPLICATIONS: The data open up avenues for the development of a unique class of therapeutic agents that act by targeting RelA and other aberrantly active proteins to nucleoli, thus killing cancer cells.

High-throughput sequencing of Fasciola spp. shows co-infection and intermediate forms in Balochistan, but only Fasciola gigantica in the Punjab province of Pakistan.

Fasciola gigantica and Fasciola hepatica are digenetic trematodes causing fasciolosis in ruminants. The host and geographical distribution of both Fasciola species are influenced by environmental and climatic conditions favouring survival and development of free-living stages and intermediate hosts, and livestock management practices. The aim of the present study was to describe the host distribution of the two Fasciola species in buffalo, cattle, goats, and sheep in the Balochistan and Punjab provinces of Pakistan. 359 flukes were collected from a total of 32 livers from the four livestock species. Deep amplicon sequencing of the internal transcribed spacer region 2 of ribosomal DNA (rDNA ITS-2) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND-1) loci confirmed co-infection of F. hepatica and F. gigantica in Balochistan and single species F. gigantica infection in Punjab. In Balochistan, co-infections and hybrids of both Fasciola species were identified in cattle, with more F. hepatica detected than F. gigantica. However, F. hepatica was the only species identified in goats, and F. gigantica was the only species identified in buffalo. In Punjab, all flukes were confirmed as F. gigantica in each of the four livestock species. Overall, the results indicate differences in the host and geographical distribution of F. gigantica and F. hepatica, and provide useful knowledge for the development of control strategies for livestock and humans.

Seeing around corners: Cells solve mazes and respond at a distance using attractant breakdown.

During development and metastasis, cells migrate large distances through complex environments. Migration is often guided by chemotaxis, but simple chemoattractant gradients between a source and sink cannot direct cells over such ranges. We describe how self-generated gradients, created by cells locally degrading attractant, allow single cells to navigate long, tortuous paths and make accurate choices between live channels and dead ends. This allows cells to solve complex mazes efficiently. Cells' accuracy at finding live channels was determined by attractant diffusivity, cell speed, and path complexity. Manipulating these parameters directed cells in mathematically predictable ways; specific combinations can even actively misdirect them. We propose that the length and complexity of many long-range migratory processes, including inflammation and germ cell migration, means that self-generated gradients are needed for successful navigation.

Generation of a conditional CREB Ser133Ala knockin mouse.

Phosphorylation of Ser133 in the transcription factor CREB is an important mechanism for regulating its transcriptional activity, however recent work has suggested significant roles for other regulatory inputs into CREB. To allow study of this in vivo, we have generated a Ser133 to alanine knockin mutation in the mouse CREB locus. As CREB knockout is perinatal lethal, a minigene strategy was used to allow conditional knockin of the Ser133Ala mutation in adult mice using Cre recombinase. While some expression of the mutated protein was observed prior to Cre expression, following Cre expression in either T cells or neurons only the mutated CREB protein was detected.

N-WASP Control of LPAR1 Trafficking Establishes Response to Self-Generated LPA Gradients to Promote Pancreatic Cancer Cell Metastasis.

Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.

Accepting from the best donor; analysis of long-lifetime donor fluorescent protein pairings to optimise dynamic FLIM-based FRET experiments.

FRET biosensors have proven very useful tools for studying the activation of specific signalling pathways in living cells. Most biosensors designed to date have been predicated on fluorescent protein pairs that were identified by, and for use in, intensity based measurements, however fluorescence lifetime provides a more reliable measurement of FRET. Both the technology and fluorescent proteins available for FRET have moved on dramatically in the last decade. Lifetime imaging systems have become increasingly accessible and user-friendly, and there is an entire field of biology dedicated to refining and adapting different characteristics of existing and novel fluorescent proteins. This growing pool of fluorescent proteins includes the long-lifetime green and cyan fluorescent proteins Clover and mTurquoise2, the red variant mRuby2, and the dark acceptor sREACh. Here, we have tested these donors and acceptors in appropriate combinations against the standard or recommended norms (EGFP and mTFP as donors, mCherry and either Ypet or Venus as acceptors) to determine if they could provide more reliable, reproducible and quantifiable FLIM-FRET data to improve on the dynamic range compared to other donors and breadth of application of biosensor technologies. These tests were performed for comparison on both a wide-field, frequency domain system and a multiphoton, TCSPC time domain FLIM system. Clover proved to be an excellent donor with extended dynamic range in combination with mCherry on both platforms, while mRuby2 showed a high degree of variability and poor FRET efficiencies in all cases. mTFP-Venus was the most consistent cyan-yellow pair between the two FLIM methodologies, but mTurquoise2 has better dynamic range and transfers energy consistently over time to the dark acceptor sRCh. Combination of mTFP-sRCh with Clover-mCherry would allow the simultaneous use of two FLIM-FRET biosensors within one sample by eliminating the crosstalk between the yellow acceptor and green donor emissions.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.

MSK1 regulates transcriptional induction of Arc/Arg3.1 in response to neurotrophins.

The immediate early gene activity-regulated cytoskeletal protein (Arc)/Arg3.1 and the neurotrophin brain-derived neurotrophic factor (BDNF) play important roles in synaptic plasticity and learning and memory in the mammalian brain. However, the mechanisms by which BDNF regulates the expression of Arc/Arg3.1 are unclear. In this study, we show that BDNF acts via the ERK1/2 pathway to activate the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 then induces Arc/Arg3.1 expression via the phosphorylation of histone H3 at the Arc/Arg3.1 promoter. MSK1 can also phosphorylate the transcription factor cyclic-AMP response element-binding protein (CREB) on Ser133. However, this is not required for BDNF-induced Arc.Arg3.1 transcription as a Ser133Ala knockin mutation had no effect on Arc/Arg3.1 induction. In parallel, ERK1/2 directly activates Arc/Arg3.1 mRNA transcription via at least one serum response element on the promoter, which bind a complex of the Serum Response Factor (SRF) and a Ternary Complex Factor (TCF).

Twin mole and viable fetus: The case for misdiagnosis.

OBJECTIVE: To report the Sheffield Trophoblast Centre experience of twin molar gestations and review this in the light of international experience. CASE: Thirty patients with possible twin molar gestations were registered from 1986 to 2004 (during which period 7,200 cases of mole were seen). The accuracy of suspected clinical and histologic diagnoses was investigated. RESULTS: In 10 cases twin mole/fetus had been suspected clinically but not confirmed when products of conception were examined. In 3 of these cases the pregnancy had been therapeutically terminated because of clinical (ultrasound) suspicion of coexisting molar pregnancy. In the 19 cases where twin mole/fetus was suspected, central histopathology review was possible in 14 cases. Only 7 were confirmed. In 2 further cases twin molar gestation was diagnosed on specimens referred for central review as partial mole singleton pregnancies. For confirmed cases the pregnancy outcome was term delivery in 5 cases and miscarriage in 4. CONCLUSION: Clinical and histopathologic diagnosis of twin molar pregnancies is inaccurate in many suspected cases; therefore, a second (expert) opinion should be sought. When the diagnosis is accurate, maternal and fetal complications are common. However, in suspected cases the pregnancy may be allowed to proceed, with caution, if the mother wishes.

An elevated neutrophil-lymphocyte ratio is associated with adverse outcomes following single time-point paracetamol (acetaminophen) overdose: a time-course analysis.

BACKGROUND: The innate immune system is profoundly dysregulated in paracetamol (acetaminophen)-induced liver injury. The neutrophil-lymphocyte ratio (NLR) is a simple bedside index with prognostic value in a number of inflammatory conditions. AIM: To evaluate the prognostic accuracy of the NLR in patients with significant liver injury following single time-point and staggered paracetamol overdoses. PATIENTS AND METHODS: Time-course analysis of 100 single time-point and 50 staggered paracetamol overdoses admitted to a tertiary liver centre. Timed laboratory samples were correlated with time elapsed after overdose or admission, respectively, and the NLR was calculated. RESULTS: A total of 49/100 single time-point patients developed hepatic encephalopathy (HE). Median NLRs were higher at both 72 (P=0.0047) and 96 h after overdose (P=0.0041) in single time-point patients who died or were transplanted. Maximum NLR values by 96 h were associated with increasing HE grade (P=0.0005). An NLR of more than 16.7 during the first 96 h following overdose was independently associated with the development of HE [odds ratio 5.65 (95% confidence interval 1.67-19.13), P=0.005]. Maximum NLR values by 96 h were strongly associated with the requirement for intracranial pressure monitoring (P<0.0001), renal replacement therapy (P=0.0002) and inotropic support (P=0.0005). In contrast, in the staggered overdose cohort, the NLR was not associated with adverse outcomes or death/transplantation either at admission or subsequently. CONCLUSION: The NLR is a simple test which is strongly associated with adverse outcomes following single time-point, but not staggered, paracetamol overdoses. Future studies should assess the value of incorporating the NLR into existing prognostic and triage indices of single time-point paracetamol overdose.

A molecular toggle after exocytosis sequesters the presynaptic syntaxin1a molecules involved in prior vesicle fusion.

Neuronal synapses are among the most scrutinized of cellular systems, serving as a model for all membrane trafficking studies. Despite this, synaptic biology has proven difficult to interrogate directly in situ due to the small size and dynamic nature of central synapses and the molecules within them. Here we determine the spatial and temporal interaction status of presynaptic proteins, imaging large cohorts of single molecules inside active synapses. Measuring rapid interaction dynamics during synaptic depolarization identified the small number of syntaxin1a and munc18-1 protein molecules required to support synaptic vesicle exocytosis. After vesicle fusion and subsequent SNARE complex disassembly, a prompt switch in syntaxin1a and munc18-1-binding mode, regulated by charge alteration on the syntaxin1a N-terminal, sequesters monomeric syntaxin1a from other disassembled fusion complex components, preventing ectopic SNARE complex formation, readying the synapse for subsequent rounds of neurotransmission.

Selective kinase inhibitors as tools for neuroscience research.

Signal transduction cascades, including the MAPK, PI3 kinase, Ca(2+) and PKC pathways, play important roles in neurons downstream of multiple signals including neurotrophins and neurotransmitters. Small molecule kinase inhibitors that block these pathways provide a powerful way of studying the in vivo or cellular roles of these signaling systems. Over the last 15 years there has been a major effort by the pharmaceutical industry to develop kinase inhibitors as potential drugs for a variety of diseases including cancer and auto-immunity. As a result of this there are now many compounds available that can be used as research tools. One major drawback is however that many of these compounds are not truly selective for a single kinase, and therefore the possibility that their cellular effects may be due to an off target activity must be considered. This problem has been brought into sharp relief by modern in vitro screening methods that allow an inhibitor to be screened against a significant proportion of the kinome. In this review we discuss the advantages and problems with the use of kinase inhibitors as research tools and describe some of the available compounds that target pathways important to neurons.

Secretory vesicles are preferentially targeted to areas of low molecular SNARE density.

Intercellular communication is commonly mediated by the regulated fusion, or exocytosis, of vesicles with the cell surface. SNARE (soluble N-ethymaleimide sensitive factor attachment protein receptor) proteins are the catalytic core of the secretory machinery, driving vesicle and plasma membrane merger. Plasma membrane SNAREs (tSNAREs) are proposed to reside in dense clusters containing many molecules, thus providing a concentrated reservoir to promote membrane fusion. However, biophysical experiments suggest that a small number of SNAREs are sufficient to drive a single fusion event. Here we show, using molecular imaging, that the majority of tSNARE molecules are spatially separated from secretory vesicles. Furthermore, the motilities of the individual tSNAREs are constrained in membrane micro-domains, maintaining a non-random molecular distribution and limiting the maximum number of molecules encountered by secretory vesicles. Together our results provide a new model for the molecular mechanism of regulated exocytosis and demonstrate the exquisite organization of the plasma membrane at the level of individual molecular machines.