Alice D. Lam

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational “opening” of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.

Structural and Functional Analysis of Tomosyn Identifies Domains Important in Exocytotic Regulation

Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a β-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K+-induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.

Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.

Silent hippocampal seizures and spikes identified by foramen ovale electrodes in Alzheimer's disease

We directly assessed mesial temporal activity using intracranial foramen ovale electrodes in two patients with Alzheimers disease (AD) without a history or EEG evidence of seizures. We detected clinically silent hippocampal seizures and epileptiform spikes during sleep, a period when these abnormalities were most likely to interfere with memory consolidation. The findings in these index cases support a model in which early development of occult hippocampal hyperexcitability may contribute to the pathogenesis of AD.

Differential interaction of tomosyn with syntaxin and SNAP25 depends on domains in the WD40 β-propeller core and determines its inhibitory activity.

Background: Tomosyns WD40 domain affects its ability to inhibit exocytosis. Results: Unstructured loops in the WD40 domain are involved in tomosyns diffusion and organization on the plasma membrane. Conclusion: These key loops mediate tomosyns binding to the SNARE protein SNAP25. Significance: Novel findings regarding tomosyns membranal distribution and interactions shed new light on regulation of exocytosis by the SNARE complex and tomosyn. Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyns activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyns dynamics at the PM and its relation to its inhibition of exocytosis.

Mapping dynamic protein interactions to insulin secretory granule behavior with TIRF-FRET.

Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells.

A Combined Optogenetic-Knockdown Strategy Reveals a Major Role of Tomosyn in Mossy Fiber Synaptic Plasticity

Neurotransmitter release probability (P(r)) largely determines the dynamic properties of synapses. While much is known about the role of presynaptic proteins in transmitter release, their specific contribution to synaptic plasticity is unclear. One such protein, tomosyn, is believed to reduce P(r) by interfering with the SNARE complex formation. Tomosyn is enriched at hippocampal mossy fiber-to-CA3 pyramidal cell synapses (MF-CA3), which characteristically exhibit low P(r), strong synaptic facilitation, and pre-synaptic protein kinase A (PKA)-dependent long-term potentiation (LTP). To evaluate tomosyns role in MF-CA3 function, we used a combined knockdown (KD)-optogenetic strategy whereby presynaptic neurons with reduced tomosyn levels were selectively activated by light. Using this approach in mouse hippocampal slices, we found that facilitation, LTP, and PKA-induced potentiation were significantly impaired at tomosyn-deficient synapses. These findings not only indicate that tomosyn is a key regulator of MF-CA3 plasticity but also highlight the power of a combined KD-optogenetic approach to determine the role of presynaptic proteins.

Medication prescribing and patient-reported outcome measures in people with epilepsy in Bhutan

OBJECTIVE The aim of this study was to assess medication prescribing and patient-reported outcomes among people with epilepsy (PWE) in Bhutan and introduce criteria for evaluating unmet epilepsy care needs, particularly in resource-limited settings. METHODS People with epilepsy in Bhutan (National Referral Hospital, 2014-2015) completed a questionnaire, the Quality of Life in Epilepsy Inventory (QOLIE-31), and an electroencephalogram (EEG). Management gap was the proportion of participants meeting any of six prespecified criteria based on best practices and the National Institute for Health and Care Excellence (NICE) guidelines. RESULTS Among 253 participants (53% female, median: 24years), 93% (n=235) were treated with antiepileptic drugs (AEDs). Seventy-two percent (n=183) had active epilepsy (≥1 seizure in the prior year). At least one criterion was met by 55% (n=138) of participants, whereas the treatment gap encompassed only 5% (n=13). The criteria were the following: 1. Among 18 participants taking no AED, 72% (n=13) had active epilepsy. 2. Among 26 adults on subtherapeutic monotherapy, 46% (n=12) had active epilepsy. 3. Among 48 participants reporting staring spells, 56% (n=27) were treated with carbamazepine or phenytoin. 4. Among 101 female participants aged 14-40years, 23% (n=23) were treated with sodium valproate. 5. Among 67 participants reporting seizure-related injuries, 87% (n=58) had active epilepsy. 6. Among 111 participants with a QOLIE-31 score below 50/100, 77% (n=86) had active epilepsy. Years since first AED treatment (odds ratio: 1.07, 95% CI: 1.03, 1.12) and epileptiform discharges on EEG (odds ratio: 1.95, 95% CI: 1.15, 3.29) were significantly associated with more criteria met. CONCLUSIONS By defining the management gap, subpopulations at greatest need for targeted interventions may be prioritized, including those already taking AEDs.

Validation of a smartphone-based EEG among people with epilepsy: A prospective study

Our objective was to assess the ability of a smartphone-based electroencephalography (EEG) application, the Smartphone Brain Scanner-2 (SBS2), to detect epileptiform abnormalities compared to standard clinical EEG. The SBS2 system consists of an Android tablet wirelessly connected to a 14-electrode EasyCap headset (cost ~ 300 USD). SBS2 and standard EEG were performed in people with suspected epilepsy in Bhutan (2014–2015), and recordings were interpreted by neurologists. Among 205 participants (54% female, median age 24 years), epileptiform discharges were detected on 14% of SBS2 and 25% of standard EEGs. The SBS2 had 39.2% sensitivity (95% confidence interval (CI) 25.8%, 53.9%) and 94.8% specificity (95% CI 90.0%, 97.7%) for epileptiform discharges with positive and negative predictive values of 0.71 (95% CI 0.51, 0.87) and 0.82 (95% CI 0.76, 0.89) respectively. 31% of focal and 82% of generalized abnormalities were identified on SBS2 recordings. Cohen’s kappa (κ) for the SBS2 EEG and standard EEG for the epileptiform versus non-epileptiform outcome was κ = 0.40 (95% CI 0.25, 0.55). No safety or tolerability concerns were reported. Despite limitations in sensitivity, the SBS2 may become a viable supportive test for the capture of epileptiform abnormalities, and extend EEG access to new, especially resource-limited, populations at a reduced cost.

Eastern Equine Encephalitis Treated With Intravenous Immunoglobulins

We report the case of a 68-year-old man from southeastern Massachusetts presenting with encephalitis due to eastern equine encephalitis (EEE) virus. Despite the high morbidity and mortality rate of EEE, the patient made a near complete recovery in the setting of receiving early intravenous immunoglobulins.