Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder.

Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies.

Aß vaccination in combination with behavioral enrichment in aged beagles: effects on cognition, Aß, and microhemorrhages.

Beta-amyloid (Aß) immunotherapy is a promising intervention to slow Alzheimer's disease. Aging dogs naturally accumulate Aß and show cognitive decline. An active vaccine against fibrillar Aß 1-42 (VAC) in aged beagles resulted in maintenance but not improvement of cognition along with reduced brain Aß. Behavioral enrichment (ENR) led to cognitive benefits but no reduction in Aß. We hypothesized cognitive outcomes could be improved by combining VAC with ENR in aged dogs. Aged dogs (11-12 years) were placed into 4 groups: (1) control/control (C/C); (2) control/VAC (C/V); (3) ENR/control (E/C); and (4) ENR/VAC (E/V) and treated for 20 months. VAC decreased brain Aß, pyroglutamate Aß, increased cerebrospinal fluid Aß 42 and brain-derived neurotrophic factor RNA levels but also increased microhemorrhages. ENR reduced brain Aß and prevented microhemorrhages. The combination treatment resulted in a significant maintenance of learning over time, reduced Aß, and increased brain-derived neurotrophic factor mRNA despite increased microhemorrhages; however, there were no benefits to memory. These results suggest that the combination of immunotherapy with behavioral enrichment leads to cognitive maintenance associated with reduced neuropathology that may benefit people with Alzheimer's disease.

The active-zone protein Munc13 controls the use-dependence of presynaptic voltage-gated calcium channels.

Presynaptic calcium channel function is critical for converting electrical information into chemical communication but the molecules in the active zone that sculpt this function are poorly understood. We show that Munc13, an active-zone protein essential for exocytosis, also controls presynaptic voltage-gated calcium channel (VGCC) function dictating their behavior during various forms of activity. We demonstrate that in vitro Munc13 interacts with voltage-VGCCs via a pair of basic residues in Munc13's C2B domain. We show that elimination of this interaction by either removal of Munc13 or replacement of Munc13 with a Munc13 C2B mutant alters synaptic VGCC's response to and recovery from high-frequency action potential bursts and alters calcium influx from single action potential stimuli. These studies illustrate a novel form of synaptic modulation and show that Munc13 is poised to profoundly impact information transfer at nerve terminals by controlling both vesicle priming and the trigger for exocytosis.

Activity-driven local ATP synthesis is required for synaptic function.

Cognitive function is tightly related to metabolic state, but the locus of this control is not well understood. Synapses are thought to present large ATP demands; however, it is unclear how fuel availability and electrical activity impact synaptic ATP levels and how ATP availability controls synaptic function. We developed a quantitative genetically encoded optical reporter of presynaptic ATP, Syn-ATP, and find that electrical activity imposes large metabolic demands that are met via activity-driven control of both glycolysis and mitochondrial function. We discovered that the primary source of activity-driven metabolic demand is the synaptic vesicle cycle. In metabolically intact synapses, activity-driven ATP synthesis is well matched to the energetic needs of synaptic function, which, at steady state, results in ∼10(6) free ATPs per nerve terminal. Despite this large reservoir of ATP, we find that several key aspects of presynaptic function are severely impaired following even brief interruptions in activity-stimulated ATP synthesis.

Roles for ca(2+) mobilization and its regulation in mast cell functions.

Mobilization of Ca(2+) in response to IgE receptor-mediated signaling is a key process in many aspects of mast cell function. Here we summarize our current understanding of the molecular bases for this process and the roles that it plays in physiologically relevant mast cell biology. Activation of IgE receptor signaling by antigen that crosslinks these complexes initiates Ca(2+) mobilization as a fast wave that is frequently followed by a series of Ca(2+) oscillations which are dependent on Ca(2+) influx-mediated by coupling of the endoplasmic reticulum luminal Ca(2+) sensor STIM1 to the calcium release activated calcium channel protein Orai1. Granule exocytosis depends on this process, together with the activation of protein kinase C isoforms, and specific roles for these signaling steps are beginning to be understood. Ca(2+) mobilization also plays important roles in stimulated exocytosis of recycling endosomes and newly synthesized cytokines, as well as in antigen-mediated chemotaxis of rat mucosal mast cells. Phosphoinositide metabolism plays key roles in all of these processes, and we highlight these roles in several cases.

Stimulated association of STIM1 and Orai1 is regulated by the balance of PtdIns(4,5)P2 between distinct membrane pools.

We have previously shown that PIP5KIß and PIP5KIγ generate functionally distinct pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] important for antigen-stimulated Ca(2+) entry in mast cells. In the present study, we find that association of the endoplasmic reticulum (ER) Ca(2+) sensor, STIM1, and the store-operated Ca(2+) channel, Orai1, stimulated by thapsigargin-mediated ER store depletion, is enhanced by overexpression of PIP5KIß and inhibited by overexpression of PIP5KIγ. These different PIP5KI isoforms cause differential enhancement of PtdIns(4,5)P(2) in detergent-resistant membrane (DRM) fractions, which comprise ordered lipid regions, and detergent-solubilized membrane (DSM) fractions, which comprise disordered lipid regions. Consistent with these results, the inositol 5-phosphatase L10-Inp54p, which is targeted to ordered lipids, decreases PtdIns(4,5)P(2) in the DRM fraction and inhibits thapsigargin-stimulated STIM1-Orai1 association and store-operated Ca(2+) entry, whereas the inositol 5-phosphatase S15-Inp54p, which is targeted to disordered lipids, decreases PtdIns(4,5)P(2) in the DSM fraction and enhances STIM1-Orai1 association. Removal of either the STIM1 C-terminal polylysine sequence (amino acids 677-685) or an N-terminal polyarginine sequence in Orai1 (amino acids 28-33) eliminates this differential sensitivity of STIM1-Orai1 association to PtdIns(4,5)P(2) in the distinctive membrane domains. Our results are consistent with a model of PtdIns(4,5)P(2) balance, in which store-depletion-stimulated STIM1-Orai1 association is positively regulated by the ordered lipid pool of PtdIns(4,5)P(2) and negatively regulated by PtdIns(4,5)P(2) in disordered lipid domains.

A basic sequence in STIM1 promotes Ca2+ influx by interacting with the C-terminal acidic coiled coil of Orai1.

Store-operated Ca(2+) entry (SOCE) is a ubiquitous signaling process in eukaryotic cells in which the endoplasmic reticulum (ER)-localized Ca(2+) sensor, STIM1, activates the plasma membrane-localized Ca(2+) release-activated Ca(2+) (CRAC) channel, Orai1, in response to emptying of ER Ca(2+) stores. In efforts to understand this activation mechanism, we recently identified an acidic coiled-coil region in the C-terminus of Orai1 that contributes to physical association between these two proteins, as measured by fluorescence resonance energy transfer, and is necessary for Ca(2+) influx, as measured by an intracellular Ca(2+) indicator. Here, we present evidence that a positively charged sequence of STIM1 in its CRAC channel activating domain, human residues 384-386, is necessary for activation of SOCE, most likely because this sequence interacts directly with the acidic coiled coil of Orai1 to gate Ca(2+) influx. We find that mutation to remove positive charges in these residues in STIM1 prevents its stimulated association with wild-type Orai1. However, association does occur between this mutant STIM1 and Orai1 that is mutated to remove negative charges in its C-terminal coiled coil, indicating that other structural features are sufficient for this interaction. Despite this physical association, we find that thapsigargin fails to activate SOCE following coexpression of mutant STIM1 with either wild type or mutant Orai1, implicating STIM1 residues 384-386 in transmission of the Ca(2+) gating signal to Orai1 following store depletion.

Molecular clustering of STIM1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+ stores and on electrostatic interactions.

Activation of store operated Ca(2+) entry involves stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER), and calcium channel subunit (Orai1/CRACM1), localized to the plasma membrane. Confocal microscopy shows that thapsigargin-mediated depletion of ER Ca(2+) stores in RBL mast cells causes a redistribution of STIM1, labeled with monomeric red fluorescent protein (mRFP), to micrometer-scale ER-plasma membrane junctions that contain Orai1/CRACM1, labeled with monomeric Aequorea coerulescens green fluorescent protein (AcGFP). Using fluorescence resonance energy transfer (FRET), we determine that this visualized coredistribution is accompanied by nanoscale interaction of STIM1-mRFP and AcGFP-Orai1/CRACM1. We find that antigen stimulation of immunoglobulin E receptors causes much less Orai1/CRACM1 and STIM1 association, but strong interaction is observed under conditions that prevent refilling of ER stores. Stimulated association monitored by FRET is inhibited by sphingosine derivatives in parallel with inhibition of Ca(2+) influx. Similar structural and functional effects are caused by mutation of acidic residues in the cytoplasmic segment of Orai1/CRACM1, suggesting a role for electrostatic interactions via these residues in the coupling of Orai1/CRACM1 to STIM1. Our results reveal dynamic molecular interactions between STIM1 and Orai1/CRACM1 that depend quantitatively on electrostatic interactions and on the extent of store depletion.

Optimized fluorescent trimethoprim derivatives for in vivo protein labeling.

The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.

Orientation and alkylation effects on cation-pi interactions in aqueous solution.

We have investigated the orientation dependence of the cation-pi interaction between a phenyl ring and a pyridinium ring in the context of a flexible model system in water. Of the four possible positions of the pyridinium nitrogen, ipso, ortho, meta, and para, we found a variation in the interaction energy of about 0.75 kcal mol(-1), with the stacking of the ipso-pyridinium ring providing the strongest interaction. The observed stability is attributed to the maximization of the electrostatic interaction, minimization of rotamers, and possible differences in hydration phenomena arising from alkylation.