Synaptic nanomodules underlie the organization and plasticity of spine synapses.

Experience results in long-lasting changes in dendritic spine size, yet how the molecular architecture of the synapse responds to plasticity remains poorly understood. Here a combined approach of multicolor stimulated emission depletion microscopy (STED) and confocal imaging in rat and mouse demonstrates that structural plasticity is linked to the addition of unitary synaptic nanomodules to spines. Spine synapses in vivo and in vitro contain discrete and aligned subdiffraction modules of pre- and postsynaptic proteins whose number scales linearly with spine size. Live-cell time-lapse super-resolution imaging reveals that NMDA receptor-dependent increases in spine size are accompanied both by enhanced mobility of pre- and postsynaptic modules that remain aligned with each other and by a coordinated increase in the number of nanomodules. These findings suggest a simplified model for experience-dependent structural plasticity relying on an unexpectedly modular nanomolecular architecture of synaptic proteins

Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons

Defects in synapse structure and function precede motor neuron degeneration in Drosophila models of FUS-related ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis associated with motor neuron degeneration and muscular atrophy. One gene associated with ALS encodes the DNA/RNA-binding protein Fused in Sarcoma (FUS). There now exist two Drosophila models of ALS. In one, human FUS with ALS-causing mutations is expressed in fly motor neurons; in the other, the gene cabeza (caz), the fly homolog of FUS, is ablated. These FUS-ALS flies exhibit larval locomotor defects indicative of neuromuscular dysfunction and early death. The locus and site of initiation of this neuromuscular dysfunction remain unclear. We show here that in FUS-ALS flies, motor neuron cell bodies fire action potentials that propagate along the axon and voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild-type and FUS-ALS motor neurons. In marked contrast, the amplitude of synaptic currents evoked in the postsynaptic muscle cell is decreased by \u3e80% in FUS-ALS larvae. Furthermore, the frequency but not unitary amplitude of spontaneous miniature synaptic currents is decreased dramatically in FUS-ALS flies, consistent with a change in quantal content but not quantal size. Although standard confocal microscopic analysis of the larval neuromuscular junction reveals no gross abnormalities, superresolution stimulated emission depletion (STED) microscopy demonstrates that the presynaptic active zone protein bruchpilot is aberrantly organized in FUS-ALS larvae. The results are consistent with the idea that defects in presynaptic terminal structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS

Therapeutic targeting of cathepsin C::from pathophysiology to treatment

Cathepsin C (CatC) is a highly conserved tetrameric lysosomal cysteine dipeptidyl aminopeptidase. The best characterized physiological function of CatC is the activation of pro-inflammatory granule-associated serine proteases. These proteases are synthesized as inactive zymogens containing an N-terminal pro-dipeptide, which maintains the zymogen in its inactive conformation and prevents premature activation, which is potentially toxic to the cell. The activation of serine protease zymogens occurs through cleavage of the N-terminal dipeptide by CatC during cell maturation in the bone marrow. In vivo data suggest that pharmacological inhibition of pro-inflammatory serine proteases would suppress or attenuate deleterious effects of inflammatory/auto-immune disorders mediated by these proteases. The pathological deficiency in CatC is associated with Papillon-Lefèvre syndrome. The patients however do not present marked immunodeficiency despite the absence of active serine proteases in immune defense cells. Hence, the transitory pharmacological blockade of CatC activity in the precursor cells of the bone marrow may represent an attractive therapeutic strategy to regulate activity of serine proteases in inflammatory and immunologic conditions. A variety of CatC inhibitors have been developed both by pharmaceutical companies and academic investigators, some of which are currently being employed and evaluated in preclinical/clinical trials

Glucose decouples intracellular Ca2+ activity from glucagon secretion in mouse pancreatic islet alpha-cells.

The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca(2+)](i)) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca(2+)](i) and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K(ATP) channel activity but not on tetrodotoxin-sensitive Na(+) channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca(2+)](i) and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K(ATP) channel activity or reduction in α-cell [Ca(2+)](i). Our results demonstrate that glucose uncouples the positive relationship between [Ca(2+)](i) and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway

Effects of voltage-gated sodium channel inhibition on islet [Ca²⁺]<i>_i</i> and hormone secretion.

<p>Gray and black traces represent α- and β-cells, respectively. A, representative intracellular calcium responses to tetrodotoxin (TTX) in an intact mouse islet perifused at 1 mM glucose. Increasing concentrations of TTX were perifused at times indicated by the arrows. TTX stimulates calcium activity in α-cells while having no noticeable effects on β-cells. Fluo-4 intensity is expressed in arbitrary units. The figure is representative of 18 α-cells analyzed from 6 islets harvested from 3 mice. B, effects of TTX on glucagon and insulin secretion from intact perifused islets. Isolated islets were exposed to 1 mM glucose for 30 minutes (from −30 to 0 min). Glucagon and insulin responses were measured for 9 minutes at 1 mM glucose (G1), and then TTX was perifused. Experiment was repeated 6 times, 900 islets from 12 mice were used. Error bars represent the standard error of the mean.</p

Effects of pharmacological modulation of K_ATP channels on islet [Ca²⁺]<i>_i</i> and hormone secretion.

<p>Gray and black traces represent α- and β-cells, respectively. A, representative intracellular calcium responses to K_ATP channel activation by 100 µM diazoxide from an islet perifused at 1 mM (G1). Fluo-4 intensity is expressed in arbitrary units. The figure is representative of 25 α-cells analyzed from 7 islets harvested from 3 mice. B, Effect of diazoxide on hormone secretion from intact perifused islets. Isolated islets were exposed to 1 mM glucose for 30 minutes (from -30 to 0 min). Glucagon and insulin responses were measured for 12 minutes at 1 mM glucose, and diazoxide was perifused for 30 minutes at 100 µM. Experiment was repeated 3 times, 450 islets from 6 mice were used. Error bars represent the standard error of the mean. C, D, and E, representative Fluo-4 responses to K_ATP channel inhibition from an islet perifused at 1 mM glucose. The figure shows 3 different α-cells from the same islet exposed to 100 µM tolbutamide, and is representative of 34 α-cells analyzed from 10 islets harvested from 3 mice. F, glucagon and insulin responses were measured for 9 minutes at 1 mM glucose, and then tolbutamide was perifused at 100 µM. Experiment was repeated 6 times, 900 islets from 12 mice were used. G, representative Fluo-4 responses to 100 µM tolbutamide (TTX) in an intact islet perifused at 12 mM. H, glucagon and insulin secretion from islets perifused at 1 mM glucose and 100 µM tolbutamide for 15 minutes, then glucose concentration was increased to 12 mM. Experiment was repeated 3 times, 450 islets from 6 mice were used.</p

Effects of high-voltage-gated calcium channel inactivation on islet [Ca²⁺]<i>_i</i> and hormone secretion.

<p>A, representative intracellular calcium responses to blockade of N- and L-type calcium channels. Gray traces represent α-cell [Ca²⁺]<i>_i</i>, and black traces indicate β-cell [Ca²⁺]<i>_i</i>. Fluo-4 intensity is expressed in arbitrary units. Calcium responses from two α-cells in the same islet are shown. N- and L-type channel inhibitors (1 µM ω-conotoxin and 20 µM nifedipine, respectively) were perifused. The figure is representative of 15 α-cells from 5 islets isolated from 3 mice. B, increasing concentrations of nifedipine were perifused at times indicated by the arrows. Nifedipine (≤ 10 µM) reduces calcium activity in α-cells without affecting β-cell [Ca²⁺]<i>_i</i>. The figure is representative of 10 α-cells from 3 islets harvested from 3 mice. C, effects of nifedipine on hormone secretion from intact perifused islets. Islets were exposed to 1 mM glucose for 30 minutes (from −30 to 0 min). Glucagon and insulin responses (gray and black traces, respectively) were measured for 15 minutes at 1 mM glucose (G1), and 20 µM nifedipine was added to the perifusion medium for 30 minutes. Experiment was repeated 4 times, 600 islets from 8 mice were used. Error bars represent the standard error of the mean.</p

Glucose effects on hormone secretion from perifused islets.

<p>Isolated islets were exposed to 1 mM glucose for 30 minutes (from −30 to 0 min). Then, both glucagon and insulin responses (gray and black traces, respectively) were measured for 15 minutes at 1 mM glucose (G1). The perifusion was changed to 12 mM (G12) for 15 minutes, and then switched back to 1 mM. Experiment was repeated 3 times, 450 islets from 6 mice were used. Error bars represent the standard error of the mean. To compare the volume of islets with different diameters and volumes, individual islets were mathematically converted to standard islet equivalents (IEQs) with a diameter of 150 µm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047084#pone.0047084-Ricordi1" target="_blank">[44]</a>.</p

Effects of L-arginine on islet NAD(P)H, [Ca²⁺]<i>_i</i>, and hormone secretion.

<p>Gray and black columns and traces represent α- and β-cells, respectively. A, arginine-dependent NAD(P)H responses from intact islets. Islets were perifused at 1 mM glucose (G1) and exposed to step-increases in arginine concentration. Data are normalized to minimal and maximal β-cell NAD(P)H obtained with FCCP and cyanide, respectively. The α-cell NAD(P)H changes to arginine (Arg) were statistically significant (<i>p</i><0.01, 32 α-cells measured from 10 islets, 3 mice) and α-cell NAD(P)H intensity was different from β-cell intensity for each condition tested (<i>p</i><0.01). Error bars indicate the standard error of the mean. B, averaged intracellular calcium responses to arginine. Data are expressed in percent change in Fluo-4 intensity compared to baseline at 1 mM glucose. α-cell responses to arginine were significant at all concentrations (<i>p</i><0.01, n = 42) and β-cell responses were significant at 10 and 20 mM arginine (<i>p</i><0.01, n = 11). C, intracellular calcium responses to 10 mM arginine from an islet perifused at 1 mM glucose (G1). 2 α-cells from the same islet are presented. Fluo-4 intensity is expressed in arbitrary units. The figure is representative of 11 α-cells from 4 islets. D, glucagon and insulin responses were measured for 9 minutes at 1 mM glucose, and then arginine was perifused at 10 mM for 18 minutes. Finally, glucose was added to the perifusion medium at 12 mM for 18 minutes. Experiment was repeated 4 times, 600 islets from 8 mice were used. Error bars represent the standard error of the mean.</p